Diaryl macrocycle polymorph

ABSTRACT

This disclosure relates to polymorphs of (7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxatriazacyclotridecin-4(5H)-one that are useful in the treatment of disease, such as cancer, in mammals. This disclosure also relates to compositions including such polymorphs, and to methods of using such compositions in the treatment of diseases, such as cancer, in mammals, especially in humans.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/188,846, filed Jul. 6, 2015, and U.S.Provisional Patent Application Ser. No. 62/218,672, filed on Sep. 15,2015, the disclosures of which are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to polymorphs of(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxatriazacyclotridecin-4(5H)-onethat are useful in the treatment of disease, such as cancer, in mammals.This disclosure also relates to compositions including such polymorphs,and to methods of using such compositions in the treatment of diseases,such as cancer, in mammals, especially in humans.

BACKGROUND

The compound(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxatriazacyclotridecin-4(5H)-one(also herein referred to as “Compound I”) represented by the formula I

is a potent small-molecule multi-target kinase inhibitor showingactivity against wild-type and mutant ALK (anaplastic lymphoma kinase),wild-type and mutant ROS1 (ROS1 proto-oncogene receptor tyrosinekinase), the TRK family of kinases (tropomyosin-related receptortyrosine kinases), JAK2 of the Janus family of kinases, SRC (Src familyof protein tyrosine kinases (SFKs)) and FAK (focal adhesion kinase).Compound I has properties, including anti-tumor properties, that arepharmacologically mediated through inhibition of tyrosine kinasereceptors. Compound I is disclosed in International Patent ApplicationNo. PCT/US2015/012597, which is incorporated herein by reference in itsentirety.

Protein kinases are key regulators for cell growth, proliferation andsurvival. A variety of diseases, such as cancer, pain, neurologicaldiseases, autoimmune diseases, and inflammation, have been shown to bemediated by receptor tyrosine kinases, such as ALK, ROS1, TRK, JAK2, SRCand FAK. For example, genetic and epigenetic alterations can accumulatein cancer cells leading to abnormal activation of signal transductionpathways which drive malignant processes. Manning, G. et al., Science2002, 298, 1912-1934. Pharmacological inhibition of these signalingpathways presents promising intervention opportunities for targetedcancer therapies. Sawyers, C., Nature 2004, 432, 294-297.

While Compound I has found application in treating disease associatedwith receptor tyrosine kinases, such as ALK, ROS1, TRK, JAK2, SRC andFAK, it is advantageous to have polymorphic forms having improvedproperties, such as improved crystallinity, dissolution properties,and/or decreased hygroscopicity, while maintaining chemical andenantiomeric stability properties.

SUMMARY

In one aspect, the present disclosure provides a crystalline form of(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxa-triazacyclotridecin-4(5H)-one.

In another embodiment, the crystalline polymorph form of(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxa-triazacyclotridecin-4(5H)-oneis anhydrous.

In another embodiment, the crystalline polymorph form 1 of compound Ican be represented by the formula

In a further embodiment, the crystalline polymorph form has a powderX-ray diffraction pattern comprising a peak at diffraction angle (2θ) of27.4±0.1. In a further embodiment, the crystalline polymorph form has apowder X-ray diffraction pattern comprising peaks at diffraction angles(2θ) of 9.4±0.1 and 27.4±0.1. In a further embodiment, the crystallinepolymorph form has a powder X-ray diffraction pattern comprising peaksat diffraction angles (2θ) of 9.4±0.1, 18.8±0.1, and 27.4±0.1. In afurther embodiment, the crystalline polymorph form has a powder X-raydiffraction pattern comprising peaks at diffraction angles (2θ) of9.4±0.1, 16.5±0.1, 18.8±0.1, and 27.4±0.1. In a further embodiment, thecrystalline polymorph form has a powder X-ray diffraction patterncomprising peaks at diffraction angles (2θ) of 9.4±0.1, 16.5±0.1,18.8±0.1, 22.8±0.1, and 27.4±0.1. In a further embodiment, thecrystalline polymorph form has a powder X-ray diffraction patterncomprising peaks at diffraction angles (2θ) of 9.4±0.1, 16.1±0.1,16.5±0.1, 18.8±0.1, 22.8±0.1, and 27.4±0.1. In a further embodiment, thecrystalline polymorph form has a powder X-ray diffraction patterncomprising peaks at diffraction angles (2θ) of 9.4±0.1, 16.1±0.1,16.5±0.1, 18.8±0.1, 21.2±0.1, 22.8±0.1, and 27.4±0.1.

In a further aspect the crystalline form has a powder X-ray diffractionpattern comprising peaks at diffraction angles (2θ) essentially the sameas shown in FIG. 1.

The present disclosure further provides a pharmaceutical compositioncomprising a polymorph form 1 of Compound I of the formula

The present disclosure further provides a capsule comprisingpharmaceutical compositions as described herein.

In another aspect, the disclosure provides a method of treating disease,especially cancer, in a mammal, including a human, the method comprisingadministering to the mammal a therapeutically effective amount ofpolymorph form 1 of(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxa-triazacyclotridecin-4(5H)-one,as described herein, or a pharmaceutical composition comprisingpolymorph form 1 of(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxa-triazacyclotridecin-4(5H)-one,as described herein.

In one embodiment, the present disclosure provides a method of treatingabnormal cell growth in a mammal, including a human, in need of suchtreatment comprising, administering to said mammal a therapeuticallyeffective amount of the free base polymorph form 1 of Compound I. Inanother embodiment, the abnormal cell growth is mediated by at least onegenetically altered tyrosine kinase.

In another embodiment, the abnormal cell growth is mediated by ALK,ROS1, TRK, JAK2, SRC, FAK or a combination thereof. In anotherembodiment, the abnormal cell growth is mediated by wild-type of mutantALK. In another embodiment, the abnormal cell growth is mediated bywild-type of mutant ROS1. In another embodiment, the abnormal cellgrowth is mediated by wild-type of mutant TRK. In another embodiment,the abnormal cell growth is mediated by wild-type of mutant JAK2. Inanother embodiment, the abnormal cell growth is mediated by wild-type ofmutant SRC. In another embodiment, the abnormal cell growth is mediatedby wild-type of mutant FAK.

In another embodiment, the abnormal cell growth is cancer. In anotherembodiment, the cancer is selected from the group consisting of lungcancer, non-small cell lung cancer, small cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck,hepatocellular carcinoma, cutaneous or intraocular melanoma, uterinecancer, ovarian cancer, rectal cancer, cancer of the anal region,stomach cancer, colon cancer, breast cancer, carcinoma of the fallopiantubes, carcinoma of the endometrium, carcinoma of the cervix, carcinomaof the vagina, carcinoma of the vulva, Hodgkin's Disease, gastric andesophago-gastric cancers, cancer of the endocrine system, cancer of thethyroid gland, cancer of the parathyroid gland, cancer of the adrenalgland, sarcoma of soft tissue, cancer of the urethra, cancer of thepenis, prostate cancer, chronic or acute leukemia, lymphocyticlymphomas, such as anaplastic large cell lymphoma, cancer of thebladder, cancer of the kidney or ureter, renal cell carcinoma, carcinomaof the renal pelvis, neoplasms of the central nervous system (CNS),glioblastoma, primary CNS lymphoma, spinal axis tumors, brain stemglioma, pituitary adenoma, inflammatory myofibroblastic tumors, andcombinations thereof.

In another aspect, the disclosure provides a compound of the formula II

wherein R¹ and R² are each independently H or PG, and R³ and R⁴ are eachindependently C₁-C₄ alkyl.

In another aspect, the disclosure provides processes for preparing acompound of the formula B

Additional embodiments, features, and advantages of the disclosure willbe apparent from the following detailed description and through practiceof the disclosure. The compounds of the present disclosure can bedescribed as embodiments in any of the following enumerated clauses. Itwill be understood that any of the embodiments described herein can beused in connection with any other embodiments described herein to theextent that the embodiments do not contradict one another.

1. A crystalline polymorph form of(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxatriazacyclotridecin-4(5H)-one.

2. The crystalline polymorph form of clause 1, wherein the crystallineform is a polymorph form of the free base of(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxatriazacyclotridecin-4(5H)-one.

3. The crystalline polymorph form of clause 1 or 2, wherein thecrystalline polymorph form has a powder X-ray diffraction patterncomprising a peak at diffraction angle (2θ) of 27.4±0.1.

4. The crystalline polymorph form of any one of clauses 1 to 3, whereinthe crystalline polymorph form has a powder X-ray diffraction patterncomprising peaks at diffraction angles (2θ) of 9.4±0.1 and 27.4±0.1.

5. The crystalline polymorph form of any one of clauses 1 to 4, whereinthe crystalline polymorph form has a powder X-ray diffraction patterncomprising peaks at diffraction angles (2θ) of 9.4±0.1, 18.8±0.1, and27.4±0.1.

6. The crystalline polymorph form of any one of clauses 1 to 5, whereinthe crystalline polymorph form has a powder X-ray diffraction patterncomprising peaks at diffraction angles (2θ) of 9.4±0.1, 16.5±0.1,18.8±0.1, and 27.4±0.1.

7. The crystalline polymorph form of any one of clauses 1 to 6, whereinthe crystalline polymorph form has a powder X-ray diffraction patterncomprising peaks at diffraction angles (2θ) of 9.4±0.1, 16.5±0.1,18.8±0.1, 22.8±0.1, and 27.4±0.1.

8. The crystalline polymorph form of any one of clauses 1 to 7, whereinthe crystalline polymorph form has a powder X-ray diffraction patterncomprising peaks at diffraction angles (2θ) of 9.4±0.1, 16.1±0.1,16.5±0.1, 18.8±0.1, 22.8±0.1, and 27.4±0.1.

9. The crystalline polymorph form of any one of clauses 1 to 8, whereinthe crystalline polymorph form has a powder X-ray diffraction patterncomprising peaks at diffraction angles (2θ) of 9.4±0.1, 16.1±0.1,16.5±0.1, 18.8±0.1, 21.2±0.1, 22.8±0.1, and 27.4±0.1.

10. A crystalline polymorph form of(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxatriazacyclotridecin-4(5H)-onehaving a powder X-ray diffraction pattern substantially the same asshown in FIG. 1.

11. A pharmaceutical composition comprising the crystalline polymorphform of any one of the preceding claims.

12. A method of treating disease in a mammal, the method comprisingadministering to the mammal a therapeutically effective amount of thecrystalline polymorph form of any one of clauses 1 to 10.

13. The method of clause 12, wherein the mammal is a human.

14. The method of clause 12 or 13, wherein the disease is selected fromthe group consisting of cancer, pain, neurological diseases, autoimmunediseases, and inflammation.

15. The method of any one of clauses 12 to 14, wherein the disease iscancer.

16. The method of clause 15, wherein the cancer is selected from thegroups consisting of lung cancer, non-small cell lung cancer, small celllung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of thehead or neck, hepatocellular carcinoma, cutaneous or intraocularmelanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of theanal region, stomach cancer, colon cancer, breast cancer, carcinoma ofthe fallopian tubes, carcinoma of the endometrium, carcinoma of thecervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin'sDisease, gastric and esophago-gastric cancers, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, chronic or acuteleukemia, lymphocytic lymphomas, such as anaplastic large cell lymphoma,cancer of the bladder, cancer of the kidney or ureter, renal cellcarcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), glioblastoma, primary CNS lymphoma, spinal axistumors, brain stem glioma, pituitary adenoma, inflammatorymyofibroblastic tumors, and combinations thereof.

17. The method of clause 16, wherein the cancer is non-small cell lungcancer.

18. A compound of the formula II

wherein R¹ and R² are each independently H or PG, and R³ and R⁴ are eachindependently a C₁-C₄ alkyl.

19. The compound of clause 18, wherein IV and R² are PG.

20. The compound of clause 18, wherein R² is H.

21. The compound of clause 18 or 19, wherein R¹ is H.

22. The compound of clause 18 or 20, wherein R¹ is PG.

23. The compound of clause 18 or 21, wherein R² is PG.

24. The compound of any one of clauses 18 to 23, wherein R³ and R⁴ aremethyl.

25. The compound of any one of clauses 18 to 24, wherein PG is selectedfrom the group consisting of FMOC, Boc, Cbz, Ac, trifluoroacetyl,phthalimide, Bn, trityl, benzylidene, and Ts.

26. The compound of any one of clauses 18 to 25, wherein PG is Boc.

27. A compound of the formula B-14

28. A process for preparing a compound of the formula I

comprising

a. contacting a compound of the formula A

with a compound of the formula B-14

in the presence of a base to provide a compound of the formula C

or

b. contacting a compound of the formula C with an inorganic base toprovide a compound of the formula D

or

c. contacting a compound of the formula D with an acid to provide acompound of the formula E

or

d. contacting a compound of the formula E with a base in the presence ofa phosphinate reagent to provide the compound of the formula I.

29. A process for preparing a compound of the formula B

wherein

PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and

R³ and R⁴ are each independently C₁-C₄ alkyl;

comprising

a. contacting a compound of the formula B-1

wherein R⁴ is C₁-C₄ alkyl; with a compound of the formula B-2R

wherein R³ is C₁-C₄ alkyl, and PG is selected from the group consistingof FMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts; in the presence of an azodicarboxylate reagent anda phosphine reagent to provide a compound of the formula B-3

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; or

b. contacting a compound of the formula B-3

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; with(R)-2-methyl-2-propanesulfinamide to provide a compound of the formulaB-5

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; or

c. contacting a compound of the formula B-5

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; with a reducing agent toprovide a compound of the formula B-6

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; or

d. contacting a compound of the formula B-6

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; with an iodine reagent toprovide a compound of the formula B.

30. A process for preparing a compound of the formula B

wherein

PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and

R³ and R⁴ are each independently C₁-C₄ alkyl;

comprising

a. reacting a compound of the formula B-7

wherein R⁴ is C₁-C₄ alkyl; under conditions suitable for preparing acompound of the formula B-8

wherein R⁴ is C₁-C₄ alkyl; and PG is selected from the group consistingof FMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts; or

b. contacting a compound of the formula B-8

wherein R⁴ is C₁-C₄ alkyl; and PG is selected from the group consistingof FMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts; with a compound of the formula B-2R

wherein R³ is C₁-C₄ alkyl, and PG is selected from the group consistingof FMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts; in the presence of an azodicarboxylate reagent anda phosphine reagent to provide a compound of the formula B-9

wherein each PG is independently selected from the group consisting ofFMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts, provided that each PG is different; and R³ and R⁴are each independently C₁-C₄ alkyl; or

c. contacting a compound of the formula B-9

wherein each PG is independently selected from the group consisting ofFMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts, provided that each PG is different; and R³ and R⁴are each independently C₁-C₄ alkyl; with an inorganic base to provide acompound of the formula B.

31. A process for preparing a compound of the formula B

wherein

PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and

R³ and R⁴ are each independently C₁-C₄ alkyl;

comprising

a. reacting a compound of the formula B-10

with a compound of the formula B-2S

wherein R³ is C₁-C₄ alkyl, and PG is selected from the group consistingof FMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts; in the presence of a base to provide a compound ofthe formula B-11

wherein R³ is C₁-C₄ alkyl, and PG is selected from the group consistingof FMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts; or

b. contacting a compound of the formula B-11

wherein R³ is C₁-C₄ alkyl, and PG is selected from the group consistingof FMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts; with a nucleophile to provide a compound of theformula B-12

wherein R³ and R⁴ are each independently C₁-C₄ alkyl, and PG is selectedfrom the group consisting of FMOC, Boc, Cbz, Ac, trifluoroacetyl,phthalimide, Bn, trityl, benzylidene, and Ts; or

c. contacting a compound of the formula B-12

wherein R³ and R⁴ are each independently C₁-C₄ alkyl, and PG is selectedfrom the group consisting of FMOC, Boc, Cbz, Ac, trifluoroacetyl,phthalimide, Bn, trityl, benzylidene, and Ts; with a reducing agent toprovide a compound of the formula B.

30. A process for preparing a compound of the formula B

wherein

PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and

R³ and R⁴ are each independently C₁-C₄ alkyl;

comprising

a. reacting a compound of the formula B-12

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; R³ and R⁴are each independently C₁-C₄ alkyl; under conditions suitable forpreparing a compound of the formula B-13

wherein each PG is independently selected from the group consisting ofFMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts, provided that each PG is different; R³ is C₁-C₄alkyl; and R⁵ is C₁-C₃ alkyl; or

b. contacting a compound of the formula B-13

wherein each PG is independently selected from the group consisting ofFMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts, provided that each PG is different; R³ is C₁-C₄alkyl; and R⁵ is C₁-C₃ alkyl; with a reducing agent to provide acompound of the formula B-9

wherein each PG is independently selected from the group consisting ofFMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts, provided that each PG is different; and R³ and R⁴are each independently C₁-C₄ alkyl; or

c. contacting a compound of the formula B-9

wherein each PG is independently selected from the group consisting ofFMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts, provided that each PG is different; and R³ and R⁴are each independently C₁-C₄ alkyl; with an inorganic base to provide acompound of the formula B.

Definitions

As used herein, the term “alkyl” includes a chain of carbon atoms, whichis optionally branched and contains from 1 to 4 carbon atoms, and thelike may be referred to as “lower alkyl.” Illustrative alkyl groupsinclude, but are not limited to, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, and tert-butyl.

As used herein, unless otherwise indicated, the term “abnormal cellgrowth” refers to cell growth that is independent of normal regulatorymechanisms (e.g., loss of contact inhibition).

As used herein, unless otherwise indicated, the term “treating” meansreversing, alleviating, inhibiting the progress of (i.e. curativetreatment), or preventing the disorder or condition to which such termapplies, or one or more symptoms of such disorder or condition. The term“treatment”, as used herein, unless otherwise indicated, refers to theact of treating as “treating” as defined immediately above.“Preventative” treatment is meant to indicate a postponement ofdevelopment of a disease, a symptom of a disease, or medical condition,suppressing symptoms that may appear, or reducing the risk of developingor recurrence of a disease or symptom. “Curative” treatment includesreducing the severity of or suppressing the worsening of an existingdisease, symptom, or condition. Thus, treatment includes ameliorating orpreventing the worsening of existing disease symptoms, preventingadditional symptoms from occurring, ameliorating or preventing theunderlying systemic causes of symptoms, inhibiting the disorder ordisease, e.g., arresting the development of the disorder or disease,relieving the disorder or disease, causing regression of the disorder ordisease, relieving a condition caused by the disease or disorder, orstopping the symptoms of the disease or disorder.

The term “subject” refers to a mammalian patient in need of suchtreatment, such as a human.

As used herein, the term “essentially the same” with reference to X-raydiffraction peak positions means that typical peak position andintensity variability are taken into account. For example, one skilledin the art will appreciate that the peak positions (20) will show someinter-apparatus variability, typically as much as 0.1°. Further, oneskilled in the art will appreciate that relative peak intensities willshow inter-apparatus variability as well as variability due to degree ofcrystallinity, preferred orientation, prepared sample surface, and otherfactors known to those skilled in the art, and should be taken asqualitative measures only.

As used herein, the term “protecting group” or “PG” refers to any groupas commonly known to one of ordinary skill in the art that can beintroduced into a molecule by chemical modification of a functionalgroup, such as an amine or hydroxyl, to obtain chemoselectivity in asubsequent chemical reaction. It will be appreciated that suchprotecting groups can be subsequently removed from the functional groupat a later point in a synthesis to provide further opportunity forreaction at such functional groups or, in the case of a final product,to unmask such functional group. Protecting groups have been describedin, for example, Wuts, P. G. M., Greene, T. W., Greene, T. W., & JohnWiley & Sons. (2006). Greene's protective groups in organic synthesis.Hoboken, N.J: Wiley-Interscience. One of skill in the art will readilyappreciate the chemical process conditions under which such protectinggroups can be installed on a functional group. Suitable amine protectinggroups useful in connection with the present disclosure include, but arenot limited to, 9-Fluorenylmethyl-carbonyl (FMOC), t-butylcarbonyl(Boc), benzyloxycarbonyl (Cbz), acetyl (Ac), trifluoroacetyl,phthalimide, benzyl (Bn), triphenylmethyl (trityl, Tr), benzylidene, andp-toluenesulfonyl (tosylamide, Ts).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a powder X-ray diffraction pattern of the crystalline formof free base(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxa-triazacyclotridecin-4(5H)-one,polymorph form 1.

FIG. 2 shows a differential scanning calorimetery (DSC) thermogram ofthe crystalline form of free base(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxatriazacyclotridecin-4(5H)-one,polymorph form 1. (a) TG curve; (b) TG % curve.

DETAILED DESCRIPTION

Before the present disclosure is further described, it is to beunderstood that this disclosure is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present disclosure will be limited only by the appendedclaims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this disclosure belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entireties. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in a patent, application, or other publication thatis herein incorporated by reference, the definition set forth in thissection prevails over the definition incorporated herein by reference.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. It is further noted that the claims may be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

A unique physical form of the free base of(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxatriazacyclotridecin-4(5H)-onehas been prepared according to the methods described herein. The powderX-ray diffraction (PXRD) pattern of free base polymorph form 1 is shownin FIG. 1, with corresponding tabulated data shown in Table 1.

TABLE 1 2 θ d-value Peak Height Peak intensity (%) 9.4 0.94555 5982 50.710.9 0.80934 807 9.9 13.3 0.66425 1609 20.9 15.1 0.58566 1294 14.5 16.10.54960 3653 40.7 16.5 0.53704 4873 50.6 18.8 0.47191 4970 76.2 19.40.45746 761 9.7 19.8 0.44832 634 7.4 20.9 0.42579 1671 21.1 21.2 0.419052891 29.8 22.8 0.39036 4500 53.5 23.6 0.37762 875 9.4 23.9 0.37233 260032.5 24.5 0.36394 1307 15.5 24.8 0.35903 808 8.6 25.6 0.34853 1057 16.226.7 0.33392 842 10.6 27.4 0.32578 8974 100.0 28.6 0.31174 2804 35.229.1 0.30692 711 8.3 29.5 0.30325 961 11.4 29.9 0.29850 888 11.9 30.20.29561 976 11.2 30.7 0.29110 631 6.2 32.1 0.27908 708 4.5 32.4 0.276231200 17.2 33.9 0.26451 515 8.4 34.5 0.26005 512 8.0 36.9 0.24367 69716.1 37.6 0.23930 438 3.7 38.0 0.23675 538 7.1 38.3 0.23521 598 5.9 39.10.23058 742 12.8 39.9 0.22592 658 15.7 40.4 0.22335 908 12.5 41.80.21589 941 12.0 42.9 0.21062 582 6.4 43.4 0.20849 1309 22.9 43.90.20615 720 6.8 49.6 0.18367 658 6.2

The DSC thermogram for crystalline polymorph form 1 is shown in FIG. 2.

In one aspect, the compounds and pharmaceutical compositions of theinvention specifically target tyrosine receptor kinases, in particularALK, ROS1, TRK, JAK2, SRC or FAK. Thus, these compounds andpharmaceutical compositions can be used to prevent, reverse, slow, orinhibit the activity of one or more of these kinases. In someembodiments, methods of treatment disease mediated by one or morereceptor tyrosine kinase are described herein.

Exemplary diseases include cancer, pain, neurological diseases,autoimmune diseases, and inflammation.

In some embodiments, methods of treating cancer are described hereincomprising administering a therapeutically effective amount of acrystalline polymorph form 1 of Compound I. Cancer includes but is notlimited to lung cancer, such as non-small cell lung cancer, small celllung cancer, and the like, bone cancer, pancreatic cancer, skin cancer,cancer of the head or neck, hepatocellular carcinoma, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, colon cancer, breast cancer,carcinoma of the fallopian tubes, carcinoma of the endometrium,carcinoma of the cervix, carcinoma of the vagina, carcinoma of thevulva, Hodgkin's Disease, gastric and esophago-gastric cancers, cancerof the endocrine system, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue,cancer of the urethra, cancer of the penis, prostate cancer, chronic oracute leukemia, lymphocytic lymphomas, such as anaplastic large celllymphoma, cancer of the bladder, cancer of the kidney or ureter, renalcell carcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), such as glioblastoma, primary CNS lymphoma, spinalaxis tumors, brain stem glioma, and the like, pituitary adenoma,inflammatory myofibroblastic tumors, and combinations thereof. In otherembodiments, methods are for treating lung cancer or non-small cell lungcancer.

In some embodiments, methods of treating or preventing pain aredescribed herein comprising administering a therapeutically effectiveamount of a crystalline polymorph form 1 of Compound I. Pain includes,for example, pain from any source or etiology, including cancer pain,pain from chemotherapeutic treatment, nerve pain, pain from injury, orother sources.

In some embodiments, methods of treating autoimmune diseases aredescribed herein comprising administering a therapeutically effectiveamount of a crystalline polymorph form 1 of Compound I. Autoimmunediseases include, for example, rheumatoid arthritis, Sjogren syndrome,Type I diabetes, and lupus. Exemplary neurological diseases includeAlzheimer's Disease, Parkinson's Disease, Amyotrophic lateral sclerosis,and Huntington's disease.

In some embodiments, methods of treating inflammatory diseases ofinflammation are described herein comprising administering atherapeutically effective amount of a crystalline polymorph form 1 ofCompound I. Exemplary inflammatory diseases include atherosclerosis,allergy, and inflammation from infection or injury.

In treatment methods according to the invention, an “effective amount”means an amount or dose sufficient to generally bring about the desiredtherapeutic benefit in subjects needing such treatment. Effectiveamounts or doses of the compounds of the invention may be ascertained byroutine methods, such as modeling, dose escalation, or clinical trials,taking into account routine factors, e.g., the mode or route ofadministration or drug delivery, the pharmacokinetics of the agent, theseverity and course of the infection, the subject's health status,condition, and weight, and the judgment of the treating physician. Anexemplary dose is in the range of about from about 0.1 mg to 1 g daily,or about 1 mg to 50 mg daily, or about 50 to 250 mg daily, or about 250mg to 1 g daily. The total dosage may be given in single or divideddosage units (e.g., BID, TID, QID).

Once improvement of the patient's disease has occurred, the dose may beadjusted for preventative or maintenance treatment. For example, thedosage or the frequency of administration, or both, may be reduced as afunction of the symptoms, to a level at which the desired therapeutic orprophylactic effect is maintained. Of course, if symptoms have beenalleviated to an appropriate level, treatment may cease. Patients may,however, require intermittent treatment on a long-term basis upon anyrecurrence of symptoms. Patients may also require chronic treatment on along-term basis.

Pharmaceutical Compositions

The present disclosure also relates to pharmaceutical compositionscomprising the free base polymorph form 1 of Compound I describedherein. Pharmaceutical compositions of the present disclosure may, forexample, be in a form suitable for oral administration as a tablet,capsule, pill, powder, sustained release formulations, solution,suspension, for parenteral injection as a sterile solution, suspensionor emulsion, for topical administration as an ointment or cream or forrectal administration as a suppository. The pharmaceutical compositionmay be in unit dosage forms suitable for single administration ofprecise dosages. The pharmaceutical composition may include conventionalpharmaceutically-acceptable excipients. In addition, pharmaceuticalcompositions described herein may include other medicinal orpharmaceutical agents, carriers, adjuvants, etc.

A pharmaceutically-acceptable excipient is a substance that is non-toxicand otherwise biologically suitable for administration to a subject.Such excipients facilitate administration of the compounds describedherein and are compatible with the active ingredient. Examples ofpharmaceutically-acceptable excipients include stabilizers, lubricants,surfactants, diluents, anti-oxidants, binders, coloring agents, bulkingagents, emulsifiers, or taste-modifying agents. In preferredembodiments, pharmaceutical compositions according to the invention aresterile compositions. Pharmaceutical compositions may be prepared usingcompounding techniques known or that become available to those skilledin the art.

Sterile compositions are also contemplated by the invention, includingcompositions that are in accord with national and local regulationsgoverning such compositions.

The pharmaceutical compositions and compounds described herein may beformulated as solutions, emulsions, suspensions, or dispersions insuitable pharmaceutical solvents or carriers, or as pills, tablets,lozenges, suppositories, sachets, dragees, granules, powders, powdersfor reconstitution, or capsules along with solid carriers according toconventional methods known in the art for preparation of various dosageforms. Pharmaceutical compositions of the invention may be administeredby a suitable route of delivery, such as oral, parenteral, rectal,nasal, topical, or ocular routes, or by inhalation. In some embodiments,the compositions are formulated for intravenous or oral administration.

For oral administration, the compounds the invention may be provided ina solid form, such as a tablet or capsule, or as a solution, emulsion,or suspension. To prepare the oral compositions, the compounds of theinvention may be formulated to yield a dosage of, e.g., from about 0.1mg to 1 g daily, or about 1 mg to 50 mg daily, or about 50 to 250 mgdaily, or about 250 mg to 1 g daily. Oral tablets may include the activeingredient(s) mixed with compatible pharmaceutically acceptableexcipients such as diluents, disintegrating agents, binding agents,lubricating agents, sweetening agents, flavoring agents, coloring agentsand preservative agents. Suitable inert fillers include sodium andcalcium carbonate, sodium and calcium phosphate, lactose, starch, sugar,glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, andthe like. Exemplary liquid oral excipients include ethanol, glycerol,water, and the like. Starch, polyvinyl-pyrrolidone (PVP), sodium starchglycolate, microcrystalline cellulose, and alginic acid are exemplarydisintegrating agents. Binding agents may include starch and gelatin.The lubricating agent, if present, may be magnesium stearate, stearicacid, or talc. If desired, the tablets may be coated with a materialsuch as glyceryl monostearate or glyceryl distearate to delay absorptionin the gastrointestinal tract, or may be coated with an enteric coating.

Capsules for oral administration include hard and soft gelatin capsules.To prepare hard gelatin capsules, active ingredient(s) may be mixed witha solid, semi-solid, or liquid diluent. Soft gelatin capsules may beprepared by mixing the active ingredient with water, an oil, such aspeanut oil or olive oil, liquid paraffin, a mixture of mono anddi-glycerides of short chain fatty acids, polyethylene glycol 400, orpropylene glycol.

Liquids for oral administration may be in the form of suspensions,solutions, emulsions, or syrups, or may be lyophilized or presented as adry product for reconstitution with water or other suitable vehiclebefore use. Such liquid compositions may optionally contain:pharmaceutically-acceptable excipients such as suspending agents (forexample, sorbitol, methyl cellulose, sodium alginate, gelatin,hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel andthe like); non-aqueous vehicles, e.g., oil (for example, almond oil orfractionated coconut oil), propylene glycol, ethyl alcohol, or water;preservatives (for example, methyl or propyl p-hydroxybenzoate or sorbicacid); wetting agents such as lecithin; and, if desired, flavoring orcoloring agents.

For parenteral use, including intravenous, intramuscular,intraperitoneal, intranasal, or subcutaneous routes, the agents of theinvention may be provided in sterile aqueous solutions or suspensions,buffered to an appropriate pH and isotonicity or in parenterallyacceptable oil. Suitable aqueous vehicles include Ringer's solution andisotonic sodium chloride. Such forms may be presented in unit-dose formsuch as ampoules or disposable injection devices, in multi-dose formssuch as vials from which the appropriate dose may be withdrawn, or in asolid form or pre-concentrate that can be used to prepare an injectableformulation. Illustrative infusion doses range from about 1 to 1000μg/kg/minute of agent admixed with a pharmaceutical carrier over aperiod ranging from several minutes to several days.

For nasal, inhaled, or oral administration, the inventive pharmaceuticalcompositions may be administered using, for example, a spray formulationalso containing a suitable carrier. The inventive compositions may beformulated for rectal administration as a suppository.

For topical applications, the compounds of the present invention arepreferably formulated as creams or ointments or a similar vehiclesuitable for topical administration. For topical administration, theinventive compounds may be mixed with a pharmaceutical carrier at aconcentration of about 0.1% to about 10% of drug to vehicle. Anothermode of administering the agents of the invention may utilize a patchformulation to effect transdermal delivery

Methods of preparing various pharmaceutical compositions with a specificamount of active compound are known, or will be apparent, to thoseskilled in this art. For examples, see Remington's PharmaceuticalSciences, Mack Publishing Company, Easter, Pa., 15th Edition (1975).

Drug Combinations

The inventive compounds described herein may be used in pharmaceuticalcompositions or methods in combination with one or more additionalactive ingredients in the treatment of the diseases and disordersdescribed herein. Further additional active ingredients include othertherapeutics or agents that mitigate adverse effects of therapies forthe intended disease targets. Such combinations may serve to increaseefficacy, ameliorate other disease symptoms, decrease one or more sideeffects, or decrease the required dose of an inventive compound. Theadditional active ingredients may be administered in a separatepharmaceutical composition from a compound of the present invention ormay be included with a compound of the present invention in a singlepharmaceutical composition. The additional active ingredients may beadministered simultaneously with, prior to, or after administration of acompound of the present invention.

Combination agents include additional active ingredients are those thatare known or discovered to be effective in treating the diseases anddisorders described herein, including those active against anothertarget associated with the disease. For example, compositions andformulations of the invention, as well as methods of treatment, canfurther comprise other drugs or pharmaceuticals, e.g., other activeagents useful for treating or palliative for the target diseases orrelated symptoms or conditions. For cancer indications, additional suchagents include, but are not limited to, kinase inhibitors, such as EGFRinhibitors (e.g., erlotinib, gefitinib), Raf inhibitors (e.g.,vemurafenib), VEGFR inhibitors (e.g., sunitinib), ALK inhibitors (e.g.,crizotinib) standard chemotherapy agents such as alkylating agents,antimetabolites, anti-tumor antibiotics, topoisomerase inhibitors,platinum drugs, mitotic inhibitors, antibodies, hormone therapies, orcorticosteroids. For pain indications, suitable combination agentsinclude anti-inflammatories such as NSAIDs. The pharmaceuticalcompositions of the invention may additional comprise one or more ofsuch active agents, and methods of treatment may additionally compriseadministering an effective amount of one or more of such active agents.

Synthesis Methods

In some embodiments, the disclosure provides a process for preparing acompound of the formula I

comprising

(a) contacting a compound of the formula A

with a compound of the formula B

in the presence of a base to provide a compound of the formula C

(b) contacting a compound of the formula C with an inorganic base toprovide a compound of the formula D

(c) contacting a compound of the formula D with an acid to provide acompound of the formula E

(d) contacting a compound of the formula E with a base in the presenceof a phosphinate reagent to provide the compound of the formula I.

It will be appreciated that the present disclosure provides processesfor preparing a compound of the formula I described in the paragraphsabove, comprising more than one of the step listed in the alternative.Accordingly, the present disclosure provides a process for preparing acompound of the formula I, comprising steps (a) and (b). Alternatively,the present disclosure provides a process for preparing a compound ofthe formula I, comprising steps (b) and (c). Alternatively, the presentdisclosure provides a process for preparing a compound of the formula I,comprising steps (c) and (d). Alternatively, the present disclosureprovides a process for preparing a compound of the formula I, comprisingsteps (a), (b) and (c). Alternatively, the present disclosure provides aprocess for preparing a compound of the formula I, comprising steps (b),(c) and (d). Alternatively, the present disclosure provides a processfor preparing a compound of the formula I, comprising steps (a), (b),(c) and (d).

In the first step (a), the base can be any organic base, such as anamine base. Suitable amine bases include, but are not limited to, DIEA,TEA, tributylamine, 2,6-lutidine, 2,2,6,6-tetramethylguanidine, and thelike. In some embodiments, step (a) can be carried out in the presenceof a polar protic solvent, such as an alcohol solvent. Suitable polarprotic solvents include, but are not limited to, MeOH, EtOH, iPrOH,n-BuOH, sec-BuOH, and the like. In some embodiments, the polar proticsolvent is n-BuOH. In some embodiments, step (a) can be carried out at atemperature of from about 50° C. to about 150° C. In some embodiments,the temperature is about 120° C.

In step (b), the inorganic base can be any inorganic base, such as ahydroxide base. Suitable hydroxide bases include, but are not limitedto, sodium hydroxide, lithium hydroxide, and the like. In someembodiments, step (b) can be carried out in the presence of a polarprotic solvent, a polar aprotic solvent or a mixture thereof. Suitablepolar protic solvents include, but are not limited to, MeOH, EtOH,iPrOH, n-BuOH, sec-BuOH, H₂O, and the like. Suitable polar aproticsolvents include, but are not limited to THF, 2-methyl-THF, Et₂O, DCM,EtOAc, DMF, CH₃CN, acetone, HMPT, DMSO, and the like. In someembodiments, step (b) can be carried out in a mixture of solvents, suchas THF/MeOH/H₂O. In some embodiments, step (b) can be carried out at atemperature of from about 30° C. to about 100° C. In some embodiments,the temperature is about 70° C.

In step (c), the acid can be a strong inorganic acid, such as HCl, suchas 2M HCl. In some embodiments, the acid can be a solution of a strongacid in a polar aprotic solvent, such as Et₂O. For example, a suitableacid for use in step (c) can include 2M HCl in Et₂O. In someembodiments, step (c) can be carried out in the presence of a furtherpolar aprotic solvent. Suitable polar aprotic solvents include, but arenot limited to THF, 2-methyl-THF, Et₂O, DCM, EtOAc, DMF, CH₃CN, acetone,HMPT, DMSO, and the like. In some embodiments, the polar aprotic solventis DCM. In some embodiments, step (a) can be carried out at atemperature of from about 0° C. to about 50° C. In some embodiments, thetemperature is about 25° C.

In the first step (d), the base can be any organic base, such as anamine base. Suitable amine bases include, but are not limited to, DIEA,TEA, tributylamine, 2,6-lutidine, 2,2,6,6-tetramethylguanidine, and thelike. Suitable phosphinate reagents include those known to one of skillin the art that are useful in preparing an activated ester of acarboxylic acid, such as pentafluorophenyl diphenylphosphinate (FDPP).In some embodiments, step (d) can be carried out in the presence of afurther polar aprotic solvent or a mixture of polar aprotic solvents.Suitable polar aprotic solvents include, but are not limited to THF,2-methyl-THF, Et₂O, DCM, EtOAc, DMF, CH₃CN, acetone, HMPT, DMSO, and thelike. In some embodiments, the polar aprotic solvent is a mixture of DMFand DCM. In some embodiments, step (a) can be carried out at atemperature of from about −20° C. to about 50° C. In some embodiments,the temperature is about 0° C. to about 25° C.

In some embodiments, the disclosure provides a process (Method A) forpreparing a compound of the formula B

wherein

PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and

R³ and R⁴ are each independently C₁-C₄ alkyl;

comprising

(a) contacting a compound of the formula B-1

wherein R⁴ is C₁-C₄ alkyl; with a compound of the formula B-2R

wherein R³ is C₁-C₄ alkyl, and PG is selected from the group consistingof FMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts; in the presence of an azodicarboxylate reagent anda phosphine reagent to provide a compound of the formula B-3

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; or

(b) contacting a compound of the formula B-3

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; with(R)-2-methyl-2-propanesulfinamide to provide a compound of the formulaB-5

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; or

(c) contacting a compound of the formula B-5

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; with a reducing agent toprovide a compound of the formula B-6

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; or

(d) contacting a compound of the formula B-6

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; with an iodine reagent toprovide a compound of the formula B.

It will be appreciated that the present disclosure provides processesfor preparing a compound of the formula B according to Method A asdescribed in the paragraphs above, comprising more than one of the steplisted in the alternative. Accordingly, the present disclosure providesa process for preparing a compound of the formula B, comprising steps(a) and (b). Alternatively, the present disclosure provides a processfor preparing a compound of the formula B, comprising steps (b) and (c).Alternatively, the present disclosure provides a process for preparing acompound of the formula B, comprising steps (c) and (d). Alternatively,the present disclosure provides a process for preparing a compound ofthe formula B, comprising steps (a), (b) and (c). Alternatively, thepresent disclosure provides a process for preparing a compound of theformula B, comprising steps (b), (c) and (d). Alternatively, the presentdisclosure provides a process for preparing a compound of the formula B,comprising steps (a), (b), (c) and (d).

In step (a) of Method A, the azodicarboxylate reagent can be any suchreagent known in the art. Suitable azodicarboxylate reagents include,but are not limited to, DEAD, diisopropyl azodicarboxylate (DIAD),di-(4-chlorobenzyl)azodicarboxylate (DCAD), and the like. In step (a) ofMethod A, the phosphine reagent can be any organophosphine reagentcommonly known in the art, including, but not limited totriphenylphoshine, tributylphosphine, and the like. In some embodiments,step (a) of Method A can be carried out in the presence of a polaraprotic solvent. Suitable polar aprotic solvents include, but are notlimited to THF, 2-methyl-THF, Et₂O, DCM, EtOAc, DMF, CH₃CN, acetone,HMPT, DMSO, and the like. In some embodiments, the polar aprotic solventis DCM. In some embodiments, step (a) of Method A can be carried out ata temperature of from about −20° C. to about 50° C. In some embodiments,the temperature is about 0° C. to about 25° C. In some embodiments, R³and R⁴ are each methyl. In some embodiments, PG is Boc.

In some embodiments, a compound of the formula B-2 can be prepared bycontacting (R)-1-aminopropan-2-ol with (Boc)₂O in the presence of anamine base.

In some embodiments, step (b) of Method A can be carried out in thepresence of a Lewis acid. In some embodiments, step (b) of Method A canbe carried out in the presence of a water scavenger. In someembodiments, the Lewis acid and water scavenger can be the same reagent.In some embodiments, the Lewis acid and water scavenger can be differentreagents. Suitable Lewis acids include, but are not limited to, copper(II) sulfate, magnesium sulfate, tetraethoxytitanium,tetraisopropxytitanium, and the like. Suitable water scavengers include,but are not limited to pyridinium p-toluenesulfonate, magnesium sulfate,sodium sulfate, tetraethoxytitanium, tetraisopropxytitanium, and thelike. In some embodiments, the Lewis acid and water scavenger istetraethoxytitanium. In some embodiments, step (b) of Method A can becarried out in the presence of a polar aprotic solvent. In someembodiments, step (b) of Method A can be carried out in the presence ofa mixture of polar aprotic solvents. Suitable polar aprotic solventsinclude, but are not limited to THF, 2-methyl-THF, Et₂O, DCM, EtOAc,DMF, CH₃CN, acetone, HMPT, DMSO, and the like. In some embodiments, thepolar aprotic solvent is a mixture of THF and 2-methyl-THF. In someembodiments, step (b) of Method A can be carried out at a temperature offrom about 0° C. to about 80° C. In some embodiments, the temperature isabout 15° C. to about 65° C. In some embodiments, R³ and R⁴ are eachmethyl. In some embodiments, PG is Boc.

In step (c) of Method A, the reducing agent can be any element orcompound commonly known in the art that loses (or “donates”) an electronto another chemical species in a redox chemical reaction, including butnot limited hydride reagents, elemental hydrogen, silane reagents,Hantzsch ester reagents, and the like. Suitable reducing agents include,but are not limited to, NaBH₄, LiAlH₄, and H₂. In some embodiments, thereducing agent in step (c) of Method A is NaBH₄. In some embodiments,step (c) of Method A can be carried out in the presence of a polarorganic solvent. In some embodiments, step (c) of Method A can becarried out in the presence of a mixture of polar organic solvents. Insome embodiments, the polar organic solvent or mixture of polar organicsolvents can be polar aprotic or polar protic. Suitable polar aproticsolvents include, but are not limited to THF, 2-methyl-THF, Et₂O, DCM,EtOAc, DMF, CH₃CN, acetone, HMPT, DMSO, and the like. Suitable polarprotic solvents include, but are not limited to, MeOH, EtOH, iPrOH,n-BuOH, sec-BuOH, H₂O, and the like. In some embodiments, the polaraprotic solvent is a mixture of THF and H₂O. In some embodiments, step(c) of Method A can be carried out at a temperature of from about −78°C. to about 30° C. In some embodiments, the temperature is about −50° C.to about 25° C. In some embodiments, R³ and R⁴ are each methyl. In someembodiments, PG is Boc.

In step (d) of Method A, the iodine reagent can be any iodine reagentknown in the art useful for deprotection of a sulfinamide. In someembodiments, the iodine reagent is I₂. In some embodiments, step (d) ofMethod A can be carried out in the presence of a polar organic solvent.In some embodiments, step (d) of Method A can be carried out in thepresence of a mixture of polar organic solvents. In some embodiments,the polar organic solvent or mixture of polar organic solvents can bepolar aprotic or polar protic. Suitable polar aprotic solvents include,but are not limited to THF, 2-methyl-THF, Et₂O, DCM, EtOAc, DMF, CH₃CN,acetone, HMPT, DMSO, and the like. Suitable polar protic solventsinclude, but are not limited to, MeOH, EtOH, iPrOH, n-BuOH, sec-BuOH,H₂O, and the like. In some embodiments, the polar aprotic solvent is amixture of THF and H₂O. In some embodiments, step (d) of Method A can becarried out at a temperature of from about 0° C. to about 80° C. In someembodiments, the temperature is about 25° C. to about 60° C. In someembodiments, the temperature is about 0° C. In some embodiments, R³ andR⁴ are each methyl. In some embodiments, PG is Boc.

Alternatively, in some embodiments, the disclosure provides a process(Method B) for preparing a compound of the formula B

wherein

PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and

R³ and R⁴ are each independently C₁-C₄ alkyl;

comprising

(a) reacting a compound of the formula B-7

wherein R⁴ is C₁-C₄ alkyl; under conditions suitable for preparing acompound of the formula B-8

wherein R⁴ is C₁-C₄ alkyl; and PG is selected from the group consistingof FMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts; or

(b) contacting a compound of the formula B-8

wherein R⁴ is C₁-C₄ alkyl; and PG is selected from the group consistingof FMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts; with a compound of the formula B-2R

wherein R³ is C₁-C₄ alkyl, and PG is selected from the group consistingof FMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts; in the presence of an azodicarboxylate reagent anda phosphine reagent to provide a compound of the formula B-9

wherein each PG is independently selected from the group consisting ofFMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts, provided that each PG is different; and R³ and R⁴are each independently C₁-C₄ alkyl; or

(c) contacting a compound of the formula B-9

wherein each PG is independently selected from the group consisting ofFMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts, provided that each PG is different; and R³ and R⁴are each independently C₁-C₄ alkyl; with an inorganic base to provide acompound of the formula B.

It will be appreciated that the present disclosure provides processesfor preparing a compound of the formula B according to Method B asdescribed in the paragraphs above, comprising more than one of the steplisted in the alternative. Accordingly, the present disclosure providesa process for preparing a compound of the formula B, comprising steps(a) and (b). Alternatively, the present disclosure provides a processfor preparing a compound of the formula B, comprising steps (b) and (c).Alternatively, the present disclosure provides a process for preparing acompound of the formula B, comprising steps (a), (b) and (c).

In step (a) of Method B, a compound of the formula B-7 can be reactedunder conditions suitable to introduce an amine protecting group (orPG). It will be appreciated that such conditions are commonly known toone of skill in the art, and any such conditions compatible with thefunctionality of a compound of the formula B-7 and the remainder of theprocess described in method B can be used. Suitable protecting groups(or PG) include, but are not limited to, FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts. In someembodiments, PG is trifluoroacetyl. In some embodiments, a compound ofthe formula B-7 can be reacted under conditions suitable to introduce atrifluoroacetyl.

In some embodiments, step (a) of Method B involves contacting a compoundof the formula B-7 with trifluoroacetic anhydride in the presence of anorganic base, such as an amine base. Suitable amine bases include, butare not limited to, DIEA, TEA, tributylamine, 2,6-lutidine,2,2,6,6-tetramethylguanidine, and the like. In some embodiments, step(a) of Method B can be carried out in a polar aprotic solvent. Suitablepolar aprotic solvents include, but are not limited to THF,2-methyl-THF, Et₂O, DCM, EtOAc, DMF, CH₃CN, acetone, HMPT, DMSO, and thelike. In some embodiments, the polar aprotic solvent is DCM. In someembodiments, step (a) of Method B can be carried out at a temperature offrom about −20° C. to about 25° C. In some embodiments, the temperatureis about 0° C. In some embodiments, R⁴ is methyl. In some embodiments,PG is trifluoroacetyl.

In step (b), of Method B, the azodicarboxylate reagent can be any suchreagent known in the art. Suitable azodicarboxylate reagents include,but are not limited to, DEAD, diisopropyl azodicarboxylate (DIAD),di-(4-chlorobenzyl)azodicarboxylate (DCAD), and the like. In step (b) ofMethod B, the phosphine reagent can be any organophosphine reagentcommonly known in the art, including, but not limited totriphenylphoshine, tributylphosphine, and the like. In some embodiments,step (b) of Method B can be carried out in the presence of a polaraprotic solvent. Suitable polar aprotic solvents include, but are notlimited to THF, 2-methyl-THF, Et₂O, DCM, EtOAc, DMF, CH₃CN, acetone,HMPT, DMSO, and the like. In some embodiments, the polar aprotic solventis DCM. In some embodiments, step (b) of Method B can be carried out ata temperature of from about −20° C. to about 50° C. In some embodiments,the temperature is about 0° C. to about 25° C. In some embodiments, R³and R⁴ are each methyl. In some embodiments, one PG is Boc and one PG iftrifluoroacetyl.

In some embodiments, step (c) of Method B can be carried out underconditions suitable to remove one of the PG groups in a compound of theformula B-9, while the other PG group remains intact. In step (c) ofMethod B, the inorganic base can be any inorganic base, such as ahydroxide base. Suitable hydroxide bases include, but are not limitedto, sodium hydroxide, lithium hydroxide, and the like. In someembodiments, step (c) of Method B can be carried out in the presence ofa polar protic solvent, a polar aprotic solvent, or a mixture thereof.Suitable polar protic solvents include, but are not limited to, MeOH,EtOH, iPrOH, n-BuOH, sec-BuOH, H₂O, and the like. Suitable polar aproticsolvents include, but are not limited to THF, 2-methyl-THF, Et₂O, DCM,EtOAc, DMF, CH₃CN, acetone, HMPT, DMSO, and the like. In someembodiments, step (c) of Method B can be carried out in a mixture ofsolvents, such as THF/MeOH. In some embodiments, step (c) of Method Bcan be carried out at a temperature of from about 30° C. to about 100°C. In some embodiments, the temperature is about 50° C. In someembodiments, R³ and R⁴ are each methyl. In some embodiments, one PG isBoc and one PG if trifluoroacetyl.

Alternatively, in some embodiments, the disclosure provides a process(Method C) for preparing a compound of the formula B

wherein

PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and

R³ and R⁴ are each independently C₁-C₄ alkyl;

comprising

(a) reacting a compound of the formula B-10

with a compound of the formula B-2S

wherein R³ is C₁-C₄ alkyl, and PG is selected from the group consistingof FMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts; in the presence of a base to provide a compound ofthe formula B-11

wherein R³ is C₁-C₄ alkyl, and PG is selected from the group consistingof FMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts; or

(b) contacting a compound of the formula B-11

wherein R³ is C₁-C₄ alkyl, and PG is selected from the group consistingof FMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts; with a nucleophile to provide a compound of theformula B-12

wherein R³ and R⁴ are each independently C₁-C₄ alkyl, and PG is selectedfrom the group consisting of FMOC, Boc, Cbz, Ac, trifluoroacetyl,phthalimide, Bn, trityl, benzylidene, and Ts; or

(c) contacting a compound of the formula B-12

wherein R³ and R⁴ are each independently C₁-C₄ alkyl, and PG is selectedfrom the group consisting of FMOC, Boc, Cbz, Ac, trifluoroacetyl,phthalimide, Bn, trityl, benzylidene, and Ts; with a reducing agent toprovide a compound of the formula B.

It will be appreciated that the present disclosure provides processesfor preparing a compound of the formula B according to Method C asdescribed in the paragraphs above, comprising more than one of the steplisted in the alternative. Accordingly, the present disclosure providesa process for preparing a compound of the formula B, comprising steps(a) and (b). Alternatively, the present disclosure provides a processfor preparing a compound of the formula B, comprising steps (b) and (c).Alternatively, the present disclosure provides a process for preparing acompound of the formula B, comprising steps (a), (b) and (c).

In step (a) of Method C, the base can be any strong, non-nucleophilicbase known to one of ordinary skill in the art. Suitable strong,non-nucleophilic bases include, but are not limited to, KHMDS, potassiumtert-butoxide, lithium diisopropyl amide,1,5,7-triazabicyclo(4.4.0)dec-5-ene (TBD),7-methyl-1,5,7-triazabicyclo(4.4.0)dec-5-ene (MTBD),1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU),1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,1,3,3-tetramethylguanidine(TMG), and the like. In some embodiments, the strong, non-nucleophilicbase is KHMDS. In some embodiments, step (a) of Method C can be carriedout in the presence of a polar aprotic solvent. Suitable polar aproticsolvents include, but are not limited to THF, 2-methyl-THF, Et₂O, DCM,EtOAc, DMF, CH₃CN, acetone, HMPT, DMSO, and the like. In someembodiments, the polar aprotic solvent is THF. In some embodiments, step(a) of Method C can be carried out at a temperature of from about −20°C. to about 40° C. In some embodiments, the temperature is about 0° C.to about 25° C. In some embodiments, R³ is methyl. In some embodiments,one PG is Boc.

In step (b) of Method C, the nucleophile can be any nucleophile capableof delivering a nucleophilic carbon atom to the nitrile functionalgroup. Suitable nucleophiles include but are not limited to, alkyl metalhalide reagents, such as Grignard reagents, and organolithium reagents.In some embodiments, the nucleophile in step (b) of method C is C₁-C₄alkylMgBr. In some embodiments, the nucleophile in step (b) of method Cis MeMgBr. In some embodiments, step (b) of Method C can be carried outin the presence of a polar aprotic solvent. Suitable polar aproticsolvents include, but are not limited to THF, 2-methyl-THF, Et₂O, DCM,EtOAc, DMF, CH₃CN, acetone, HMPT, DMSO, and the like. In someembodiments, the polar aprotic solvent is THF. In some embodiments, step(b) of Method C further comprises the addition of an alcohol solvent,such as methanol or ethanol, to quench the reaction. In someembodiments, step (b) of Method C further comprises the addition astrong inorganic acid to form an imminium salt. In some embodiments, thestrong acid is an HCl ether solution. In some embodiments, step (b) ofMethod C can be carried out at a temperature of from about −80° C. toabout 40° C. In some embodiments, the temperature is about −78° C. toabout 25° C. In some embodiments, R³ and R⁴ are methyl. In someembodiments, one PG is Boc.

In step (c) of Method C, the reducing agent can be any element orcompound commonly known in the art that loses (or “donates”) an electronto another chemical species in a redox chemical reaction, including butnot limited hydride reagents, elemental hydrogen, silane reagents,Hantzsch ester reagents, and the like. Suitable reducing agents include,H₂ and Hantzsch ester. In some embodiments, it is convenient to contactthe reducing agent in the presence of a catalyst, such as an iridiumcatalyst, a ruthenium catalyst, a rhodium catalyst, a palladiumcatalyst, and the like. It will be appreciated by one of skill in theart will that the catalyst can be any catalysts system known in the artthat is capable of promoting the reduction of an imminium salt to anamine. In some embodiments, the catalyst is[Ir(COD)Cl]₂/(S,S)-f-binaphane. In some embodiments, the reducing agentin step (c) of Method C is H₂ in the presence of [Ir(COD)Cl]₂ and(S,S)-f-binaphane. In some embodiments, the H₂ is applied at from about2 atmospheres of pressure to about 15 atmospheres of pressure. In someembodiments, the H₂ is applied at about 10 atmospheres of pressure.

In some embodiments, step (c) of Method C can be carried out in thepresence of a polar protic solvent, such as an alcohol solvent. Suitablepolar protic solvents include, but are not limited to, MeOH, EtOH,iPrOH, n-BuOH, sec-BuOH, and the like. In some embodiments, the polarprotic solvent is MeOH. In some embodiments, step (c) of Method C can becarried out in the presence of a polar aprotic solvent. Suitable polaraprotic solvents include, but are not limited to THF, 2-methyl-THF,Et₂O, DCM, EtOAc, DMF, CH₃CN, acetone, HMPT, DMSO, and the like. In someembodiments, the polar aprotic solvent is DCM. In some embodiments, step(c) of Method C can be carried out in a mixture of a polar proticsolvent and a polar aprotic solvent. In some embodiments, step (c) ofMethod C can be carried out in a mixture of a DCM and MeOH.

Alternatively, in some embodiments, the disclosure provides a process(Method D) for preparing a compound of the formula B

wherein

PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and

R³ and R⁴ are each independently C₁-C₄ alkyl;

comprising

(a) reacting a compound of the formula B-12

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; R³ and R⁴are each independently C₁-C₄ alkyl; under conditions suitable forpreparing a compound of the formula B-13

wherein each PG is independently selected from the group consisting ofFMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts, provided that each PG is different; R³ is C₁-C₄alkyl; and R⁵ is C₁-C₃ alkyl; or

(b) contacting a compound of the formula B-13

wherein each PG is independently selected from the group consisting ofFMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts, provided that each PG is different; R³ is C₁-C₄alkyl; and R⁵ is C₁-C₃ alkyl; with a reducing agent to provide acompound of the formula B-9

wherein each PG is independently selected from the group consisting ofFMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts, provided that each PG is different; and R³ and R⁴are each independently C₁-C₄ alkyl; or

(c) contacting a compound of the formula B-9

wherein each PG is independently selected from the group consisting ofFMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts, provided that each PG is different; and R³ and R⁴are each independently C₁-C₄ alkyl; with an inorganic base to provide acompound of the formula B.

It will be appreciated that the present disclosure provides processesfor preparing a compound of the formula B according to Method D asdescribed in the paragraphs above, comprising more than one of the steplisted in the alternative. Accordingly, the present disclosure providesa process for preparing a compound of the formula B, comprising steps(a) and (b). Alternatively, the present disclosure provides a processfor preparing a compound of the formula B, comprising steps (b) and (c).Alternatively, the present disclosure provides a process for preparing acompound of the formula B, comprising steps (a), (b) and (c).

In some embodiments, step (a) of Method D involves contacting a compoundof the formula B-12 with trifluoroacetic anhydride in the presence of anorganic base, such as an amine base. Suitable amine bases include, butare not limited to, DIEA, TEA, tributylamine, 2,6-lutidine,2,2,6,6-tetramethylguanidine, and the like. In some embodiments, step(a) of Method D can be carried out in a polar aprotic solvent. Suitablepolar aprotic solvents include, but are not limited to THF,2-methyl-THF, Et₂O, DCM, EtOAc, DMF, CH₃CN, acetone, HMPT, DMSO, and thelike. In some embodiments, the polar aprotic solvent is DCM. In someembodiments, step (a) of Method D can be carried out at a temperature offrom about −20° C. to about 25° C. In some embodiments, the temperatureis about 0° C. In some embodiments, R⁴ is methyl. In some embodiments,PG is trifluoroacetyl.

In step (b) of Method D, the reducing agent can be any element orcompound commonly known in the art that loses (or “donates”) an electronto another chemical species in a redox chemical reaction, including butnot limited hydride reagents, elemental hydrogen, silane reagents,Hantzsch ester reagents, and the like. Suitable reducing agents include,H₂ and Hantzsch ester. In some embodiments, it is convenient to contactthe reducing agent in the presence of a catalyst, such as an iridiumcatalyst, a ruthenium catalyst, a rhodium catalyst, a palladiumcatalyst, and the like. It will be appreciated by one of skill in theart will that the catalyst can be any catalysts system known in the artthat is capable of promoting the reduction of an imminium salt to anamine. In some embodiments, the catalyst is and[((R,R)-Me-DuPHOS)-Rh—(COD)]BF₄. In some embodiments, the reducing agentin step (b) of Method D is H₂ in the presence of and[((R,R)-Me-DuPHOS)-Rh—(COD)]BF₄. In some embodiments, the H₂ is appliedat from about 2 psi to about 100 psi. In some embodiments, the H₂ isapplied at about 90 psi.

In some embodiments, step (b) of Method D can be carried out in thepresence of a polar protic solvent, such as an alcohol solvent. Suitablepolar protic solvents include, but are not limited to, MeOH, EtOH,iPrOH, n-BuOH, sec-BuOH, and the like. In some embodiments, the polarprotic solvent is MeOH. In some embodiments, step (b) of Method D can becarried out in the presence of a polar aprotic solvent. Suitable polaraprotic solvents include, but are not limited to THF, 2-methyl-THF,Et₂O, DCM, EtOAc, DMF, CH₃CN, acetone, HMPT, DMSO, and the like. In someembodiments, the polar aprotic solvent is DCM. In some embodiments, step(b) of Method D can be carried out in a mixture of a polar proticsolvent and a polar aprotic solvent. In some embodiments, step (b) ofMethod D can be carried out in a mixture of a DCM and MeOH.

In some embodiments, step (c) of Method D can be carried out underconditions suitable to remove one of the PG groups in a compound of theformula B-9, while the other PG group remains intact. In step (c) ofMethod D, the inorganic base can be any inorganic base, such as a acarbonate base or a hydroxide. Suitable hydroxide bases include, but arenot limited to, sodium hydroxide, lithium hydroxide, and the like.Suitable carbonate bases include, but are not limited to, potassiumcarbonate, sodium bicarbonate, sodium carbonate, and the like. In someembodiments, the inorganic base is potassium carbonate. In someembodiments, step (c) of Method D can be carried out in the presence ofa polar protic solvent, a polar aprotic solvent, or a mixture thereof.Suitable polar protic solvents include, but are not limited to, MeOH,EtOH, iPrOH, n-BuOH, sec-BuOH, H₂O, and the like. Suitable polar aproticsolvents include, but are not limited to THF, 2-methyl-THF, Et₂O, DCM,EtOAc, DMF, CH₃CN, acetone, HMPT, DMSO, and the like. In someembodiments, step (c) of Method D can be carried out in MeOH. In someembodiments, step (c) of Method D can be carried out at a temperature offrom about 30° C. to about 100° C. In some embodiments, the temperatureis about 50° C. In some embodiments, R³ and R⁴ are each methyl. In someembodiments, one PG is Boc and one PG if trifluoroacetyl.

EXAMPLES

The examples and preparations provided below further illustrate andexemplify particular aspects of embodiments of the disclosure. It is tobe understood that the scope of the present disclosure is not limited inany way by the scope of the following examples.

Abbreviations

The examples described herein use materials, including but not limitedto, those described by the following abbreviations known to thoseskilled in the art:

g grams eq equivalents mmol millimoles mol moles mL milliliters L literspsi pounds per square inch EtOAc or EA ethyl acetate MeCN acetonitrileDCM dichloromethane MTBE methyl tert-butyl ether DEAD diethylazodicarboxylate DIAD diisopropyl azodicarboxylate [Ir(COD)Cl]₂bis(1,5-cyclooctadiene)di-μ- chlorodiiridium(I)[((R,R)—Me—DuPHOS)—Rh—(COD)]BF₄1,2-Bis((2R,5R)-2,5-dimethylphospholano)-benzene(cyclooctadiene)rhodium(I) tetrafluoroborate MHz megahertz δchemical shift THF tetrahydrofuran PE petroleum ether Rf retardationfactor DMSO-d₆ deuterated dimethyl sulfoxide CDCl₃ deuterated chloroformn-BuOH n-butanol DIEA or DIPEA or Hunig's Base n,n-diisopropylethylamineTEA triethylamine KHMDS potassium bis(trimethylsilyl)amide TMSCltrimethylsilyl chloride mm or mins minute or minutes hrs, hr or h houror hours TLC thin layer chromatography M molar MS mass spectrum m/zmass-to-charge ratio FDPP pentafluorophenyl diphenylphosphinate DMAP4-(dimethylamino)pyridine DMF N,N-dimethylformamide IPC ion-pairchromatography LCMS liquid chromatography mass spectrometry

Synthesis of Crystalline Polymorph Form 1 of Compound I

Compound I was prepared according to the following synthetic scheme:

Example 1: Preparation of5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (1) Step 1: Preparationof ethyl 5-oxo-4H-pyrazolo[1,5-a]pyrimidine-3-carboxylate (1-2)

To a mixture of ethyl 5-amino-1H-pyrazole-4-carboxylate (Sigma-Aldrich,150.00 g, 1.08 mmol) and ethyl (E)-3-ethoxyprop-2-enoate (Sigma-Aldrich,292.16 g, 2.03 mol) in DMF (3.2 L) was added Cs₂CO₃ (656.77 g, 2.02 mol)in one portion at 20° C. under N2. The mixture was stirred at 110° C.for 6 h. TLC (PE:EtOAc=1:1) showed the reaction was completed. Themixture was cooled to 20° C. and filtered through a celite pad. Thefilter cake was washed with ethyl acetate (3×30 mL). The filtrate wasadded to H₂O (2 L) and acidified with HOAc to pH=4. The resultantprecipitate was filtered to afford 1-2 (173.00 g, 834.98 mmol, 86.36%yield) as a white solid: ¹H NMR (400 MHz, DMSO-d₆) δ 8.54 (d, J=7.91 Hz,1H), 8.12 (s, 1H), 6.13 (d, J=7.91 Hz, 1H), 4.27 (q, J=7.11 Hz, 2H),1.28 (t, J=7.09 Hz, 3H).

Step 2: Preparation of 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate(1)

To a mixture of 1-2 (158.00 g, 762.59 mmol) in MeCN (1.6 L) was addedPOCl₃ (584.64 g, 3.81 mol) at 20° C. under N2. The mixture was stirredat 100° C. for 2 h. TLC (PE:EA=1:1) showed the reaction was completed.The mixture was cooled to 20° C. and poured into ice-water (5000 mL) inportions at 0° C. and stirred for 20 min. The precipitate was filteredand dried to afford 1 (110.00 g, 487.52 mmol, 63.93% yield) as a whitesolid: ¹H NMR (400 MHz, DMSO-d₆) δ 9.33 (d, J=7.28 Hz, 1H), 8.66 (s,1H), 7.41 (d, J=7.15 Hz, 1H), 4.31 (q, J=7.15 Hz, 2H), 1.32 (t, J=7.09Hz, 3H).

Example 2: Preparation of (R)-2-(1-aminoethyl)-4-fluorophenol (2) Step1: Preparation of(R)-N-(5-fluoro-2-hydroxybenzylidene)-2-methylpropane-2-sulfinamide(2-2)

To a solution of (R)-2-methylpropane-2-sulfinamide (Sigma-Aldrich,150.00 g, 1.24 mol, 1.00 eq.) and 5-fluoro-2-hydroxybenzaldehyde (2-1)(Sigma-Aldrich, 173.74 g, 1.24 mol, 1.00 eq.) in DCM (2.00 L) was addedCs₂CO₃ (646.43 g, 1.98 mol, 1.60 eq.). The mixture was stirred at 16° C.for 16 hours. TLC (PE:EtOAc=5:1) showed the reaction was completed. Thereaction mixture was quenched by addition of H₂O (1000 mL) at 0° C. andthen extracted with EtOAc (500 mL×4). The combined organic layers werewashed with brine (1000 mL) and dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give 2-2 (230.00 g, 945.33 mmol,76.24% yield). ¹HNMR (CDCl₃, 400 MHz) δ 8.64 (s, 1H), 7.22-7.11 (m, 2H),7.03-6.95 (m, 1H), 1.28 (s, 9H).

Step 2: Preparation of(R)-N-((R)-1-(5-fluoro-2-hydroxyphenyl)ethyl)-2-methylpropane-2-sulfinamide(2-3R)

To a solution of(R)-N-(5-fluoro-2-hydroxybenzylidene)-2-methylpropane-2-sulfinamide(2-2) (200.00 g, 822.03 mmol, 1.00 eq.) in THF (2.5 L) was added MeMgBr(490.09 g, 4.11 mol, 5.00 eq.) drop-wise at −65° C. under N2 over aperiod of 30 min. The mixture was then warmed to ambient temperature andstirred for 18 hours. TLC (PE:EtOAc=1:1) showed the reaction wascomplete with the production of two diastereomers. The reaction mixturewas quenched by addition of H₂O (2 L) at 0° C., the mixture wasextracted with EtOAc (500 mL×3). The combined organic layers were washedwith brine (500 mL), dried over Na₂SO₄, filtered and concentrated underreduced pressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=50/1 to 1:1) to give(R)-N-((R)-1-(5-fluoro-2-hydroxyphenyl)ethyl)-2-methylpropane-2-sulfinamide(2-3R) (125 g, the top, less polar spot with Rf: 0.5, PE:EA=1:1). ¹HNMR(CDCl₃, 400 MHz) δ: 9.17 (s, 1H), 6.68 (dd, J=3.0, 8.8 Hz, 1H), 6.47(dt, J=3.0, 8.4 Hz, 1H), 6.31 (dd, J=4.8, 8.8 Hz, 1H), 5.11 (d, J=8.0Hz, 1H), 4.28 (quin, J=7.2 Hz, 1H), 1.43 (d, J=6.8 Hz, 3H), 1.20 (s,9H).

Step 3: Preparation of (R)-2-(1-aminoethyl)-4-fluorophenol (2)

A solution of(R)-N-((R)-1-(5-fluoro-2-hydroxyphenyl)ethyl)-2-methylpropane-2-sulfinamide(2-3R) (125 g, 481.99 mmol, 1.00 eq.) in HCl/dioxane (1.5 L, 4N) wasstirred at ambient temperature for 2 hours. TLC (PE:EtOAc=2:1) showedthe reaction was complete. The mixture was filtered to give(R)-2-(1-aminoethyl)-4-fluorophenol (2) HCl salt (85 g, 443.56 mmol,90.03% yield) as a white solid. ¹HNMR (d-DMSO, 400 MHz) δ 10.24 (s, 1H),8.48 (br. s., 3H), 7.31 (dd, J=2.9, 9.7 Hz, 1H), 7.05-6.99 (m, 1H),6.98-6.93 (m, 1H), 4.59-4.45 (m, 1H), 1.46 (d, J=6.8 Hz, 3H).

Example 3: Preparation of(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxatriazacyclotridecin-4(5H)-one(Compound I) Step 1: Preparation of ethyl(R)-5-((1-(5-fluoro-2-hydroxyphenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate(3)

To a solution of (R)-2-(1-aminoethyl)-4-fluorophenol (2) (85 g, 443.56mmol, 1.00 eq.) and ethyl5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (1) (100.08 g, 443.56mmol, 1.00 eq.) in n-BuOH (2 L) was added DIEA (343.96 g, 2.66 mol, 6.00eq.). The mixture was stirred at 120° C. for 2 hrs. TLC (PE:EtOAc=1:1)showed the reaction was completed. The reaction mixture was diluted withH₂O (500 mL) at 16° C., and extracted with EtOAc (500 mL×3). Thecombined organic layers were washed with brine (500 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=10/1 to 1:3) to give ethyl(R)-5-((1-(5-fluoro-2-hydroxyphenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate(3) (122 g, 349.34 mmol, 78.76% yield, ee>99% purity) as a white solid.¹HNMR (CDCl₃, 400 MHz) δ 9.28 (br. s., 1H), 8.26 (s, 1H), 8.14 (d, J=7.5Hz, 1H), 6.95-6.89 (m, 2H), 6.87-6.80 (m, 1H), 6.18 (d, J=7.5 Hz, 1H),5.98 (d, J=8.3 Hz, 1H), 5.71-5.54 (m, 1H), 4.50-4.35 (m, 2H), 1.60 (d,J=6.8 Hz, 3H), 1.42 (t, J=7.2 Hz, 3H).

Step 2: Preparation of ethyl5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate(4)

A mixture of ethyl(R)-5-((1-(5-fluoro-2-hydroxyphenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate(3) (10.00 g, 29.04 mmol) and tert-butyl (R)-(2-hydroxypropyl)carbamate(Combi-Blocks, 7.63 g, 43.56 mmol) was azetrope dried from DCM/toluene,and then re-dissolved in DCM (11.62 mL). To the solution was added PPh₃(11.43 g, 43.56 mmol), and the mixture was stirred until the startingmaterials were completely dissolved. To the solution was added DEAD(8.81 g, 43.56 mmol) over 5 min with mixing. The reaction was stirredfor 3 hours. The reaction mixture was diluted with DCM (125 mL),followed by addition of aqueous NaOH solution (2M, 100 mL). The mixturewas stirred vigorously for 12 hours and the layers were separated. Theaqueous layer was further extracted with DCM (3×50 mL). The combinedextracts were washed with brine (50 mL), dried with Na₂SO₄, andconcentrated under reduced pressure. The residue was purified with flashchromatography (Teledyne ISCO system, silica (330 g), 0-40% ethylacetate in hexane to provide ethyl5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluorophenyl)ethyl)amino)-pyrazolo[1,5-a]pyrimidine-3-carboxylate(4) (8.88 g, 60.9% yield). LC-MS m/z 502.2 (M+H)⁺. ¹HNMR (400 MHz,CHLOROFORM-d) δ 8.24 (s, 1H), 8.21 (d, J=7.6 Hz, 1H), 7.04 (d, J=8.4 Hz,1H), 6.87 (d, J=6.0 Hz, 2H), 6.13 (d, J=7.2 Hz, 1H), 5.91 (br. s., 1H),4.58 (d, J=3.6 Hz, 1H), 4.43-4.28 (m, 2H), 3.52-3.34 (m, 2H), 1.54 (d,J=6.8 Hz, 3H), 1.47-1.36 (m, 12H), 1.30 (d, J=6.4 Hz, 3H).

Step 3: Preparation of5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylicAcid (5)

To a solution of ethyl5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate(4) (6.98 g, 13.92 mmol, 1 eq.) in methanol (65 mL) and THF (20 mL) wasadded LiOH (2M, 47.9 mL, 95.8 mmol). The mixture was heated at 70° C.for 3 hrs, cooled to ambient temperature, and then quenched with aq. HCl(2M, 95.8 mL) to adjust pH<5. The reaction mixture was extracted withCH₂Cl₂ (3×50 mL), and dried over Na₂SO₄. After filtration, evaporation,and high vacuum dry, a white solid of5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylicacid (5) was obtained which was used in the next step without furtherpurification. LC-MS m/z 474.2 (M+H)⁺.

Step 4: Preparation of5-(((R)-1-(2-(((S)-1-aminopropan-2-yl)oxy)-5-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylicAcid (6)

To a solution of5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylicacid (5) (6.59 g, 13.92 mmol) in CH₂Cl₂ (130 mL) was added HCl indioxane (4 M, 30.4 mL). Keep stirring at room temperature for 2 hoursuntil the reaction was shown to be completed by LC-MS. The reactionmixture was concentrated, and high vacuum dried to provide compound 6 asa white solid which was used in the next step without furtherpurification. LC-MS m/z 374.2 (M+H)⁺.

Step 5: Preparation of(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxatriazacyclotridecin-4(5H)-one(Compound I)

5-(((R)-1-(2-(((S)-1-aminopropan-2-yl)oxy)-5-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylicacid (6) (5.20 g, 13.93 mmol) was dissolved in DMF (75 mL) to makeSolution A. To a solution of Hunig's base (DIPEA) (14.40 g, 111.4 mmol)in DMF (150 mL) and DCM (350 mL) was added solution A (25 mL) and onethird of the total FDPP (5.62 g, 14.63 mmol) sequentially. The reactionwas stirred for 1 hour, and LC-MS showed the completion of the couplingreaction. The same process was repeated for 2 more times. The finalsolution was stirred at ambient temperature for 63 hour (or until thereaction was shown to be completed by LC-MS). The reaction was quenchedby addition of aqueous Na₂CO₃ solution (2M, 150 mL), and the mixture wasstirred for 15 min, and extracted with DCM (3×150 mL). The combinedextracts were dried with Na₂SO₄, concentrated under reduced pressure,and purified on a flash chromatography (Teledyne ISCO system, silica(220 g), 0-7.5% methanol in dichloromethane) to provide(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxatriazacyclotridecin-4(5H)-one(Compound I) (4.38 g, 12.33 mmol, 88.5% yield) as a white solid. LC-MS:m/z [M+H]⁺ 356.2. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 9.82 (dd, J=8.02, 2.29Hz, 1H), 8.81 (d, J=6.87 Hz, 1H), 8.58 (d, J=7.45 Hz, 1H), 8.04 (s, 1H),7.12 (dd, J=9.45, 3.15 Hz, 1H), 6.99-7.05 (m, 1H), 6.94-6.99 (m, 1H),6.36 (d, J=7.45 Hz, 1H), 5.53 (m, 1H), 4.45-4.52 (m, 1H), 3.90 (ddd,J=13.46, 8.31, 4.01 Hz, 1H), 3.10-3.17 (m, 1H), 1.46 (d, J=6.30 Hz, 3H),1.44 (d, J=7.45 Hz, 3H).

Example 4: Formation of Crystalline Polymorph Form 1 of Compound I

Solid Compound I (5.55 g), obtained directly from the purificationfractions of Example 3, Step 5, was re-dissolved in EA:DCM:MeOH(200:150:40), and the solution was concentrated to a volume ˜70 mL toremove most of DCM and methanol. A white crystalline solid was formed.The white crystalline solid filtered to provide a crystalline polymorphform 1 of Compound I. Anal. Calcd for C₁₈H₁₈FN₅O₂: C, 60.84; H, 5.11; N,19.71. Found: C, 60.54; H, 5.48; N, 19.88.

Large Scale Syntheses of Compound I

Example 5: Synthesis of Compound C

To a solution of A (126.40 g, 560.22 mmol, 1.00 eq.) and B-14 (175.00 g,560.22 mmol, 1.00 eq.) in n-BuOH (1.70 L) was added DIEA (485.09 g, 3.75mol, 655.53 mL, 6.70 eq.) at 25° C. The mixture was stirred at 120° C.for 5 hr. LCMS showed the starting material was completely consumed.Removed the solvent, and water (1 L) was added to the residue, and thendiluted with EtOAc (1 L), extracted with EtOAc (2 L×3). The combinedorganic layers were washed with brine (1 L), dried over Na₂SO₄, filteredand concentrated under reduced pressure to give a residue. The residuewas purified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=10/1 to 1:1) to give compound C (174.00 g, 346.92 mmol, 61.93%yield) as a white solid. LCMS: m/z 502.2 (M+H⁺). ¹H NMR (400 MHz,CHLOROFORM-d) δ 8.22 (s, 1H), 8.19 (d, J=7.6 Hz, 1H), 7.04 (d, J=8.4 Hz,1H), 6.86 (d, J=5.2 Hz, 2H), 6.14 (d, J=6.4 Hz, 1H), 6.06 (br. s., 1H),5.52 (br. s., 2H), 4.57 (d, J=3.2 Hz, 1H), 4.40-4.28 (m, 2H), 3.51-3.31(m, 2H), 1.53 (d, J=6.8 Hz, 3H), 1.47-1.32 (m, 12H), 1.29 (d, J=6.0 Hz,3H).

Example 6: Synthesis of Compound D

To a solution of5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate(C) (140.00 g, 279.13 mmol, 1 eq.) in methanol (644 mL) and THF (252 mL)was added aqueous LiOH (3.3 M, 504 mL, 1.6632 mol, 5.95 eq.). The clearsolution was heated at 70° C. for 2.5 hr. The reaction was cooled in icebath, and then quenched with aq. HCl (3.3 M, 504 mL) to adjust pH<5. Thereaction mixture was extracted with CH₂Cl₂ (1 L, and 2×500 mL). Thecombined extracts were washed with brine, and dried over Na₂SO₄. Afterfiltration, evaporation, and high vacuum dry, a white solid of5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylicacid (D) was obtained (140.78 gram). The product was used in the nextstep without further purification.

Example 7: Synthesis of Compound E

To a solution of5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylicacid (D) (132.17 g, 279.13 mmol) in CH₂Cl₂ (659 mL) was added HCl indiethyl ether (2 M, 497 mL) at ambient temperature. The reaction wasstirred at ambient temperature for 22 hours, and additional HCl diethylether solution (2M, 100 mL) was added and stirred for 5 hours. The solidproduct was filtered and washed with diethyl ether, and dried in highvacuum to provide Compound E as 3 HCl salt which was used in the nextstep directly.

Example 8: Synthesis of Compound I from Compound E

5-(((R)-1-(2-(((S)-1-aminopropan-2-yl)oxy)-5-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylicacid (E) (208 g, 557.07 mmol) (obtained from 280 gram of C) wasdissolved in DMF (1.1 L) and Hunig's Base (300 mL) to make solution A(˜1700 mL). To two 4 L reaction flasks were added DMF (608 mL), DCM(3.19 L) and Hunig's Base (500 mL), respectively. To each reaction flaskwas added solution A (160 mL) followed by the addition of FDPP (20 g,36.61 mmol). The reaction mixture was stirred for one hour and LCMSshowed the complete consumption of compound E. The same process wasrepeated until all of the solution A was added to the two reactionflasks. After the last addition, additional FDPP (10 grams) was added toeach flask and the final solutions were stirred at ambient temperatureovernight. The reaction solution from one reaction flask wasconcentrated down to 1.5 L, and then diluted with DCM (4 L) and washedwith aqueous Na₂CO₃ solution (2 M, 3 L). The aqueous layer was extractedwith DCM (3×700 mL). The combined organic layers were washed withaqueous Na₂CO₃ solution (1 M, 2 L), water (2 L) and dried with Na₂SO₄.The same work-up procedure was applied to the second reaction flask. Thecombined solutions were filtered and concentrated under reduced pressureto a volume of ˜600 mL. A large amount of precipitate was observedduring the concentration which was stirred at 0° C. for 0.5 hr and thenfiltered to provide a solid product (145 grams). The filtrate wascondensed to dryness and the residue was re-dissolved in DCM (1 L), andwashed with aqueous HCl solution (0.4 M, 500 mL), Na₂CO₃ (2M, 1 L),water (1 L), and dried with Na₂SO₄. After filtration, the solution wasconcentrated to ˜100 mL and the solid was precipitated out which wasfiltered and washed with diethyl ether (50 mL) to provide additionalproduct (10.7 gram). The combined solid was re-dissolved in 10% methanolin DCM (2 L), and filtered to get a clear solution which was furtherdiluted with methanol (500 mL). The solution was concentrated underreduced pressure to ˜400 mL and cooled at 0° C. for 1 h. The solid wasfiltered and washed with cold methanol (2×60 mL) and diethyl ether (2×75mL), and dried in high vacuum to provide(7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]-benzoxatriazacyclotridecin-4(5H)-one(Compound I) (145.302 g). The filtrate was concentrated down to ˜120 mLand then cooled at 0° C. for 30 min to provide a second crop (4.815gram). A total of 150.12 gram (75.8% yield for three steps: hydrolysis,de-boc and cyclization) of (Compound I) was obtained with a purity >98%.LC-MS: m/z [M+H]⁺ 356.2. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 9.82 (dd,J=8.02, 2.29 Hz, 1H), 8.81 (d, J=6.87 Hz, 1H), 8.58 (d, J=7.45 Hz, 1H),8.04 (s, 1H), 7.12 (dd, J=9.45, 3.15 Hz, 1H), 6.99-7.05 (m, 1H),6.94-6.99 (m, 1H), 6.36 (d, J=7.45 Hz, 1H), 5.53 (m, 1H), 4.45-4.52 (m,1H), 3.90 (ddd, J=13.46, 8.31, 4.01 Hz, 1H), 3.10-3.17 (m, 1H), 1.46 (d,J=6.30 Hz, 3H), 1.44 (d, J=7.45 Hz, 3H).

Synthesis of Compound A

Example 9: Preparation of ethyl5-oxo-4H-pyrazolo[1,5-a]pyrimidine-3-carboxylate (A-2)

3-Amino-1H-pyrazole-4-carboxylic acid ethyl ester (A-1) (Langchem Inc,2.0 kg, 12.9 mol) was charged into a 50-L jacked reactor. DMF(Industrial Grade, 20 L) was then added followed by ethyl3-ethoxyacrylate (LightChem, 3.5 kg, 24.5 mol, 1.9 eq) and Cs₂CO₃(NuoTai Chem, 8.0 kg, 24.5 mol, 1.9 eq). The reaction mixture was heatedto 110˜115° C. over 40 min and agitated at this temperature overnight.IPC by LCMS showed almost all the starting materials had been consumed.The solution was then cooled to ambient temperature over 1 hr to yield amixture. The resulting solid was collected by filtration and washed withEtOAc (6 L). The solid was collected and dissolved in water (20 L). Thesolution was then acidified with glacial acetic acid (6.5 L) to a pH 4.No exotherm was observed during the acidification. The resulting solidwas collected by filtration and washed with water (10 L). The solid wasdried at 50° C. under vacuum for 15 h to give A-2 (2.3 kg, >99.9%, 87%yield). ¹H NMR (400 MHz, DMSO-d₆) δ 8.54 (d, J=7.91 Hz, 1H), 8.12 (s,1H), 6.13 (d, J=7.91 Hz, 1H), 4.27 (q, J=7.11 Hz, 2H), 1.28 (t, J=7.09Hz, 3H).

Example 10: Preparation of5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (A)

A-2 (2.3 kg, 11.1 mol) was charged into a 50-L jacked reactor. MeCN(Industrial Grade, 23 L) was added and cooled to 15˜20° C. withagitation. Neat POCl₃ (8.5 kg, 55.5 mol, 5.0 eq) was added to themixture over 10 min with no change in reactor temperature. The reactionmixture was heated to 100˜105° C. over 1 hr and then agitated at thistemperature for 2 hr. IPC by LCMS showed that the starting material hadbeen consumed. The reaction solution was then transferred into a 100-Ljacked reactor containing ice water (50 L, 5° C.) over 1 hr. The rate ofaddition was controlled so the exothermic reaction was not allowed toexceed an internal temperature of 30° C. The resulting slurry wasagitated at 15-20° C. for 30 min. The solid that formed was filtered anddried at 45° C. under vacuum for 36 h to give A (1.8 kg, 99.9% pure byLCMS, 72% yield) as a white solid: ¹H NMR (400 MHz, DMSO-d₆) δ 9.33 (d,J=7.28 Hz, 1H), 8.66 (s, 1H), 7.41 (d, J=7.15 Hz, 1H), 4.31 (q, J=7.15Hz, 2H), 1.32 (t, J=7.09 Hz, 3H).

Synthesis of Compound B—Method A

Example 11: Preparation of tert-butyl (R)-(2-hydroxypropyl)carbamate(B-2a)

To a solution of (R)-1-aminopropan-2-ol (600.00 g, 7.99 mol, 631.58 mL,1.00 eq.) and TEA (808.33 g, 7.99 mol, 1.11 L, 1.00 eq.) in DCM (3.00 L)was added (Boc)₂O (1.74 kg, 7.99 mol, 1.84 L, 1.00 eq.). The reactionmixture was stirred at 25° C. under Na for 5 hr. TLC showed the reactionwas completed. The reaction mixture was partitioned between saturatedNaHCO₃ (500 mL) and DCM (1 L) and then washed with brine (1 L). Theorganic layer dried over anhydrous Na₂SO₄ and evaporated to givecompound B-2a (1.37 kg, 7.82 mol, 97.86% yield) as an oil. ¹H NMR (400MHz, CHLOROFORM-d) δ 5.00 (br. s., 1H), 3.89 (s, 1H), 3.25 (dd, J=2.8,10.4 Hz, 1H), 3.00 (td, J=6.8, 13.6 Hz, 1H), 2.71-2.50 (m, 1H), 1.44 (s,9H), 1.17 (d, J=6.4 Hz, 3H).

Example 12: Preparation of tert-butyl(S)-(2-(2-acetyl-4-fluorophenoxy)propyl)carbamate (B-3a)

To a solution of B-1a (500.00 g, 3.24 mol, 1.00 eq.), B-2a (851.57 g,4.86 mol, 1.50 eq.) and PPh₃ (1.27 kg, 4.86 mol, 1.50 eq.) indichloromethane (1.5 L) was added DEAD (902.79 g, 5.18 mol, 940.41 mL,1.60 eq.) drop-wise at 0° C. The solution was stirred at 25° C. for 4hours. TLC indicated one major new spot with larger polarity wasdetected, and the starting material was consumed completely. Petroleumether (1.5 L) was added to the mixture, then filtrated the solid, thesolvent of the filtrate was removed and the residue was purified bycolumn chromatography (SiO₂, Petroleum ether/Ethyl acetate=20/1 to 10:1)to give B-3a (680.00 g, 2.18 mol, 67.28% yield) as a red oil. ¹H NMR(400 MHz, CHLOROFORM-d) δ 7.38 (dd, J=3.2, 8.8 Hz, 1H), 7.13 (ddd,J=3.2, 7.2, 8.8 Hz, 1H), 6.97 (dd, J=4.0, 8.8 Hz, 1H), 5.06 (br. s.,1H), 4.63-4.52 (m, 1H), 3.52-3.39 (m, 1H), 3.38-3.27 (m, 1H), 2.59 (s,3H), 1.42 (s, 9H), 1.32 (d, J=6.4 Hz, 3H).

Example 13: Preparation of tert-butyl((S)-2-(2-((E)-1-(((R)-tert-butylsulfinyl)imino)-ethyl)-4-fluorophenoxy)propyl)carbamate(B-5a)

To a mixture of B-4 (219.98 g, 1.82 mol, 1.50 eq.), diglyme (162.35 g,1.21 mol, 172.71 mL, 1.00 eq.) and B-3a (376.00 g, 1.21 mol, 1.00 eq.)in THF (1.88 L) and 2-methyltetrahydrofuran (1.88 L) was addedtetraethoxytitanium (552.03 g, 2.42 mol, 501.85 mL, 2.00 eq.) in oneportion at 20° C. under N2. The mixture was stirred at 60° C. for 12 hr.TLC showed about 15% starting material remaining. The mixture was cooledto 20° C. Water (2 L) was added. The aqueous phase was extracted withethyl acetate (2000 mL×3). The combined organic phase was washed withsaturated brine (1 L), dried over anhydrous Na₂SO₄, filtered andconcentrated in vacuum to give a B-5a (520.00 g, crude) as a red oilwhich used for the next step without further purification.

Example 14: Preparation of tert-butyl((S)-2-(2-((R)-1-(((R)-tert-butylsulfinyl)amino)-ethyl)-4-fluorophenoxy)propyl)carbamate(B-6a)

To a solution of B-5a (520.00 g, 1.25 mol, 1.00 eq.) in THF/H₂O (3.82L/78 mL) was added NaBH₄ (142.37 g, 3.76 mol, 3.00 eq.) at −50° C., thereaction was warmed to 25° C., and then stirred at 25° C. for 12 hr. TLCshowed starting material was completely consumed. Water (1 L) was addedto the mixture and extracted with EtOAc (2 L×2). The organic layer waswashed with saturated NaCl (1 L) and dried over Na₂SO₄. Removed thesolvent and the residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=20/1 to 10:1) to give B-6a (270.00 g,570.40 mmol, 45.47% yield). ¹H NMR (400 MHz, CHLOROFORM-d) δ 7.06 (dd,J=3.2, 9.2 Hz, 1H), 6.95 (dt, J=3.2, 8.4 Hz, 1H), 6.80 (dd, J=4.4, 9.2Hz, 1H), 6.70 (br. s., 1H), 4.93 (d, J=6.0 Hz, 1H), 4.57-4.46 (m, 1H),3.68-3.65 (m, 1H), 3.59-3.57 (m, 1H), 3.22-3.10 (m, 1H), 1.47 (d, J=6.8Hz, 3H), 1.40 (s, 9H), 1.27-1.25 (m, 3H), 1.22 (s, 9H).

Example 15: Preparation of tert-butyl((S)-2-(2-((R)-1-aminoethyl)-4-fluorophenoxy)-propyl)carbamate (B-14)

To a solution of B-6a (270.00 g, 570.40 mmol, 1.00 eq.) and moleculariodine (28.95 g, 114.08 mmol, 22.98 mL, 0.20 eq.) in THF (2.16 L) wasadded H₂O (540.00 mL) at 25° C. under N2. The mixture was stirred at 50°C. for 3 hours. TLC showed the starting material was consumedcompletely. The mixture was concentrated to give B-14 (330.00 g, crude)as a white solid. LCMS: m/z 313.2 (M+H⁺). ¹H NMR (400 MHz, CHLOROFORM-d)δ 7.08 (dd, J=2.8, 9.4 Hz, 1H), 6.91-6.79 (m, 2H), 5.72 (br. s., 1H),4.55-4.32 (m, 2H), 3.52-3.41 (m, 1H), 3.31-3.19 (m, 1H), 1.42 (s, 9H),1.38 (d, J=6.8 Hz, 3H), 1.29 (d, J=6.0 Hz, 3H).

Synthesis of Compound B-14—Method B

Example 16: Preparation of(R)-2,2,2-trifluoro-N-(1-(5-fluoro-2-hydroxyphenyl)-ethyl)acetamide(B-8a)

To a solution of (R)-2-(1-aminoethyl)-4-fluorophenol hydrochloride(NetChem, 1.00 g, 5.22 mmol) and triethylamine (1.58 g, 15.66 mmol) inDCM (26.10 mL) at 0° C. was added trifluoroacetic anhydride (1.26 g,6.00 mmol) dropwise. The reaction solution was stirred for 2 hours at 0°C., and then quenched by adding to 0.5 M HCl aqueous solution (100 mL).The mixture was extracted with DCM (3×50 mL). The combined extracts werewashed with 0.5 M HCl solution (2×50 mL), water (100 mL) and brine (50mL), dried with Na₂SO₄ and concentrated under reduced pressure toprovide B-8a (1.203 g, 91.5% yield). LCMS: m/z 252 (M+H⁺).

Example 17: Preparation of tert-butyl((S)-2-(4-fluoro-2-((R)-1-(2,2,2-trifluoroacetamido)ethyl)phenoxy)propyl)carbamate(B-9a)

The mixture of(R)-2,2,2-trifluoro-N-(1-(5-fluoro-2-hydroxyphenyl)ethyl)acetamide (1.20g, 4.78 mmol) and tert-butyl (R)-(2-hydroxypropyl)carbamate (1.68 g,9.56 mmol) was azetrope dried together from DCM:Toluene. Then theresidue was re-dissolved in DCM (2.00 mL) and PPh₃ (2.57 g, 9.80 mmol)was added to the solution. The mixture was stirred until all reactantscompletely dissolved. The solution was cooled to 0° C. and DIAD (1.98 g,9.80 mmol) was added very slowly with mixing. The reaction was warmed toambient temperature and stirred for 2 hours, and then heated to 35° C.and stirred for 16 hours. The mixture was concentrated under reducedpressure and used in the next step without purification. LCMS: m/z 431(M+Na⁺).

Example 18: Preparation of tert-butyl((S)-2-(2-((R)-1-aminoethyl)-4-fluorophenoxy)propyl)carbamate (B-14)

To a solution of crude tert-butyl((S)-2-(4-fluoro-2-((R)-1-(2,2,2-trifluoroacetamido)ethyl)phenoxy)propyl)carbamate(1.95 g, 4.77 mmol) in MeOH (15 mL) and THF (5 mL) was added 2 M LiOHaqueous solution (7.03 mL). The mixture was heated at 50° C. for 6hours, cooled to ambient temperature, and diluted with water (100 mL)and 2 M NaOH solution (25 mL), and extracted with DCM (3×75 mL). Thecombined extracts were washed with 2 M NaOH solution (75 mL), dried withNa₂SO₄, concentrated under reduced pressure, and dried on high vacuum.The residue was dissolved in 1:1 DCM:Hexane (100 mL), and extracted with0.5 M HCl in 9:1 water:MeOH (3×60 mL). The combined aqueous extractswere washed with 1:3 DCM:Hexane (100 mL), neutralized with 2 M NaOHsolution (100 mL), and extracted with DCM (3×100 mL). The combined DCMextracts were dried with Na₂SO₄, concentrated under reduced pressure,and dried on high vacuum to provide a white solid B-14 (797.6 mg, 53%yield for combined three steps). LCMS: m/z 313.2 (M+H⁺). ¹H NMR (400MHz, CHLOROFORM-d) δ 7.08 (dd, J=2.8, 9.4 Hz, 1H), 6.91-6.79 (m, 2H),5.72 (br. s., 1H), 4.55-4.32 (m, 2H), 3.52-3.41 (m, 1H), 3.31-3.19 (m,1H), 1.42 (s, 9H), 1.38 (d, J=6.8 Hz, 3H), 1.29 (d, J=6.0 Hz, 3H).

Synthesis of Compound B-14—Method C

Example 19: Preparation of tert-butyl(S)-(2-(2-cyano-4-fluorophenoxy)propyl)carbamate (B-11a)

To a solution of tert-butyl (S)-(2-hydroxypropyl)carbamate (1.32 g, 7.55mmol) and 2,5-difluorobenzonitrile (Aldrich, 1.00 g, 7.19 mmol) in THF(48 mL) at 0° C. was added KHMDS (1 M, 7.55 mL). The reaction solutionwas warmed to room temperature, and stirred for 18 hours under nitrogen.The solution was concentrated, diluted with DCM (150 mL), washed with0.1 M HCl (3×150 mL), dried with Na₂SO₄, and concentrated under reducedpressure. Flash chromatography (ISCO system, silica (24 g), 0-25% ethylacetate in hexane) provided B-11a (1.43 g, 4.86 mmol, 67.58% yield).LCMS: m/z 317 (M+Na⁺). ¹H NMR (500 MHz, DMSO-d₆) δ ppm 7.72 (dd, J=8.31,3.15 Hz, 1H), 7.51-7.59 (m, 1H), 7.34 (dd, J=9.45, 4.30 Hz, 1H), 7.08(t, J=5.73 Hz, 1H), 4.58 (sxt, J=5.96 Hz, 1H), 3.15-3.25 (m, 1H),3.06-3.13 (m, 1H), 1.36 (s, 9H), 1.24 (d, J=5.73 Hz, 3H).

Example 20: Preparation of tert-butyl(S)-(2-(4-fluoro-2-(1-iminoethyl)phenoxy)propyl)-carbamate Hydrochloride(B-12a)

To a solution of tert-butyl(S)-(2-(2-cyano-4-fluorophenoxy)propyl)carbamate (100.00 mg, 0.34 mmol)in THF (1.70 mL) was added MeMgBr (3 M, 0.34 mL) at −78° C. The solutionwas warmed to room temperature and stirred for 4 hours. The reaction wasquenched with MeOH (475.20 mg, 14.83 mmol) at −78° C., then warmed toroom temperature and stirred for 2 hours. The reaction solution wasfiltered through a celite pad, concentrated to dryness under reducedpressure, and dried on high vacuum. The residue was then re-dissolved inMTBE:DCM (1:3, 5 mL), and cooled to 0° C., followed by the addition ofHCl ether solution (2 M, 0.17 mL). The reaction solution was warmed toambient temperature and stirred for 1 hour. After concentration, theresidue was suspended in MTBE (4 mL), and filtered. The solid was washedwith MTBE and dried on high vacuum to afford B-12a (62.7 mg, 52.7%yield). LCMS: m/z 334 (M+Na⁺). ¹H NMR (500 MHz, DMSO-d₆) δ ppm 12.61(bs, 1H), 11.70 (bs, 1H), 7.77 (dd, J=9.45, 3.15 Hz, 1H), 7.61-7.68 (m,1H), 7.43 (dd, J=9.45, 4.30 Hz, 1H), 7.26 (t, J=5.73 Hz, 1H), 4.68-4.76(m, 1H), 3.14-3.24 (m, 1H), 3.13-3.25 (m, 1H), 2.81 (s, 3H), 1.36 (s,9H), 1.25 (d, J=6.30 Hz, 3H).

Example 21: Preparation of tert-butyl((S)-2-(2-((R)-1-aminoethyl)-4-fluorophenoxy)-propyl)carbamateHydrochloride (B-14)

To a well-mixed solution of [Ir(COD)Cl]₂ (Strem Chemicals, 2.1 mg, 0.003mmol) and (S,S)-f-Binaphane (Strem Chemicals, 5.1 mg, 0.006 mmol) inCH₂Cl₂ (1 mL) was added tert-butyl(S)-(2-(4-fluoro-2-(1-iminoethyl)phenoxy)propyl)carbamate hydrochloride(0.3 mmol) in MeOH (2 mL). The reaction vessel is then placed into asteel autoclave. The inert atmosphere is replaced by H₂ and the reactionmixture is stirred under 10 atmospheres H₂ (150 psi) at ambienttemperature for 12 hr. The resulting mixture is concentrated undervacuum and dissolved in saturated aqueous NaHCO₃ (5 mL). After stirringfor 10 min, the mixture is extracted with CH₂Cl₂ (3×2 mL), dried overNa₂SO₄, concentrated, and dried under high vacuum to provide compoundB-14. ¹H NMR (400 MHz, CHLOROFORM-d) δ 7.08 (dd, J=2.8, 9.4 Hz, 1H),6.91-6.79 (m, 2H), 5.72 (br. s., 1H), 4.55-4.32 (m, 2H), 3.52-3.41 (m,1H), 3.31-3.19 (m, 1H), 1.42 (s, 9H), 1.38 (d, J=6.8 Hz, 3H), 1.29 (d,J=6.0 Hz, 3H).

Synthesis of Compound B-14—Method D

Example 22: Preparation of tert-butyl(S)-(2-(4-fluoro-2-(1-(2,2,2-trifluoroacetamido)-vinyl)phenoxy)propyl)carbamate(B-13a)

To a solution of tert-butyl(S)-(2-(4-fluoro-2-(1-iminoethyl)phenoxy)propyl)carbamate hydrochloride(25.00 mg, 0.072 mmol) and trifluoroacetic anhydride (17.41 mg, 0.083mmol) in DCM (0.36 mL) at 0° C. was added triethylamine (43.76 mg, 0.432mmol). The reaction solution was stirred for 3 hours at 0° C., quenchedwith addition of 0.5 M aqueous HCl (25 mL), extracted with DCM (150 mL).The extract was washed with 0.5 M aqueous HCl (25 mL), dried withNa₂SO₄, and concentrated under reduced pressure. Flash chromatography(ISCO system, silica (12 g), 0-50% ethyl acetate in hexane) providedB-13a (15.70 mg, 0.038 mmol, 53.60% yield). LCMS: m/z 429 (M+Na⁺). ¹HNMR (500 MHz, DMSO-d₆) δ ppm 10.70 (s, 1H) 7.13-7.22 (m, 1H) 7.06-7.13(m, 2H) 6.92 (t, J=6.01 Hz, 1H) 5.66 (s, 1H) 5.18 (s, 1H) 4.42 (q,J=6.30 Hz, 1H) 3.17 (dt, J=13.75, 6.01 Hz, 1H) 2.91-3.01 (m, 1H) 1.35(s, 9H) 1.13 (d, J=6.30 Hz, 3H).

Example 23: Preparation of tert-butyl((S)-2-(4-fluoro-2-((R)-1-(2,2,2-trifluoroacetamido)ethyl)phenoxy)propyl)carbamate(B-9a)

tert-butyl(S)-(2-(4-fluoro-2-(1-(2,2,2-trifluoroacetamido)vinyl)phenoxy)propyl)carbamate(3.00 mmol) and [((R,R)-Me-DuPHOS)-Rh—(COD)]BF₄ (Strem Chemicals, 0.2mol %) is placed in a glass pressure vessel, which was then purged withhydrogen for three times. Degassed methanol (10 mL) was then added andthe vessel further is purged with hydrogen and charged to 90 psihydrogen. After stirring for 20 hr the reaction mixture is evaporated toafford a residue. This residue was dissolved in EtOAc (5 mL) and thesolution was filtered through a short silica plug to remove catalystresidues. The solvent is then evaporated to afford compound B-9a.

Example 24: Preparation of tert-butyl((S)-2-(2-((R)-1-aminoethyl)-4-fluorophenoxy)propyl)carbamatehydrochloride (B-14)

To a solution of tert-butyl((S)-2-(4-fluoro-2-((R)-1-(2,2,2-trifluoroacetamido)ethyl)-phenoxy)propyl)carbamate(1.00 mmol) in methanol (30 mL) and water (10 mL) was added K₂CO₃ (3.00mmol). The mixture is heated at 60° C. until the hydrolysis wascomplete. The reaction mixture was extracted three times withdichloromethane (30 mL). The combined extracts are dried over Na₂SO₄,filtered, concentrated, and dried under high vacuum to afford compoundB-14. ¹H NMR (400 MHz, CHLOROFORM-d) δ 7.08 (dd, J=2.8, 9.4 Hz, 1H),6.91-6.79 (m, 2H), 5.72 (br. s., 1H), 4.55-4.32 (m, 2H), 3.52-3.41 (m,1H), 3.31-3.19 (m, 1H), 1.42 (s, 9H), 1.38 (d, J=6.8 Hz, 3H), 1.29 (d,J=6.0 Hz, 3H).

Testing of Crystalline Polymorph Form 1 of Compound I Example 25: PowderX-ray Diffraction (PXRD) of crystalline polymorph form 1 of

Compound I.

A sample of Compound I, crystalline polymorph form 1, was run for PXRDon a Bruker D8 Advance equipped with a 1-D Lynxeye silicon stripdetector and Cu radiation (1.54178 Å). The sample was spun duringcollection to limit preferred orientation peaks. Data was collected from2°-50° 20 using a step size of 0.02° and scan rate of 0.25 s per step.Results are shown in FIG. 1.

Example 26: Differential Scanning Calorimetry (DSC) of CrystallinePolymorph Form 1 of Compound I

DSC measurements were carried out using a Seiko Model SSC/5200Differential Scanning calorimeter. A 7.16 mg sample of Compound I,crystalline polymorph form 1, was equilibrated at 30° C., and thenramped to 380° C. at a rate of 10° C./min. The sample of Compound I,crystalline polymorph form 1, showed a melting point of 345.5° C.Results are shown in FIG. 2.

BIOLOGICAL EXAMPLES Example 27: Kinase Binding Assays

Kinase binding assays were performed at DiscoveRx using the generalKINOMEscan K_(d) Protocol (Fabian, M. A. et al., “A smallmolecule-kinase interaction map for clinical kinase inhibitors,” Nat.Biotechnol. 2005, 23(3):329-36). For most assays, kinase-tagged T7 phagestrains were prepared in an E. coli host derived from the BL21 strain.E. coli were grown to log-phase and infected with T7 phage and incubatedwith shaking at 32° C. until lysis. The lysates were centrifuged andfiltered to remove cell debris. The remaining kinases were produced inHEK-293 cells and subsequently tagged with DNA for qPCR detection.Streptavidin-coated magnetic beads were treated with biotinylated smallmolecule ligands for 30 minutes at room temperature to generate affinityresins for kinase assays. The liganded beads were blocked with excessbiotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05%Tween 20, 1 mM DTT) to remove unbound ligand and to reduce nonspecificbinding. Binding reactions were assembled by combining kinases, ligandedaffinity beads, and test compounds in 1× binding buffer (20% SeaBlock,0.17×PBS, 0.05% Tween 20, 6 mM DTT). All reactions were performed inpolystyrene 96-well plates in a final volume of 0.135 mL. The assayplates were incubated at room temperature with shaking for 1 hour andthe affinity beads were washed with wash buffer (lx PBS, 0.05% Tween20). The beads were then re-suspended in elution buffer (lx PBS, 0.05%Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated at roomtemperature with shaking for 30 minutes. The kinase concentration in theeluates was measured by qPCR. With this method, Compound I had a bindingaffinity with JAK2 of K_(d)=0.082 nM and ALK of K_(d)=5.7 nM.

Example 28: EML4-ALK B a/F3 Stable Cell Line Creation and CellProliferation Assay

The EML4-ALK wild-type gene (variant 1) was synthesized at GenScript andcloned into pCDH-CMV-MCS-EF1-Puro plasmid (System Biosciences, Inc).Ba/F3-EML4-ALK wild type cell line was generated by infecting Ba/F3cells with lentivirus containing EML4-ALK wide-type. Stable cell lineswere selected by puromycin treatment, followed by IL-3 withdrawal. 5000cells were seeded in 384 well white plate overnight before compoundtreatment. Cell proliferation was measured using CellTiter-Gloluciferase-based ATP detection assay (Promega) following themanufactures's protocol after 48 hours of various concentration ofcompound incubation. IC₅₀ determinations were performed using GraphPadPrism software (GraphPad, Inc., San Diego, Calif.). Data for Compound Iis presented in Table 2.

Example 29: Cell Proliferation Assays

Colorectal cell lines KM 12 (harboring endogenous TPM3-TRKA fusion gene)cells were cultured in DMEM medium, supplemented with 10% fetal bovineserum and 100 U/mL of penicillin/streptomycin. 5000 cells were seeded in384 well white plate for 24 hours before compounds treatment. Cellproliferation was measured using CellTiter-Glo luciferase-based ATPdetection assay (Promega) following the manufactures's protocol after 72hours incubation. IC₅₀ determinations were performed using GraphPadPrism software (GraphPad, Inc., San Diego, Calif.).

Colorectal cell line KM12 (harboring endogenous TPM3-TRKA fusion gene)cells were cultured in DMEM medium, supplemented with 10% fetal bovineserum and 100 U/mL of penicillin/streptomycin. Essential thrombocythemiacell line SET-2 cells (harboring endogenous JAK2 V618F point mutation)or T cell lymphoma Karpas-299 cell line (harboring endogenous NPM-ALKfusion gene) were cultured in RPMI medium, supplemented with 10% fetalbovine serum and 100 U/mL of penicillin/streptomycin. 5000 cells wereseeded in 384 well white plate for 24 hours before compounds treatment.Cell proliferation was measured using CellTiter-Glo luciferase-based ATPdetection assay (Promega) following the manufactures's protocol after 72hours incubation. IC₅₀ determinations were performed using GraphPadPrism software (GraphPad, Inc., San Diego, Calif.).

Data for Compound I is presented in Table 2.

TABLE 2 KM 12 SET2 Karpas 299 EML4-ALK cell prolif. cell proli. cellproli. Ba/F3 cell proli. IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) 0.5 24223.7 21.1

1-17. (canceled)
 18. A compound of the formula II

wherein R¹ and R² are each independently H or PG, and R³ and R⁴ are eachindependently C₁-C₄ alkyl.
 19. The compound of claim 18, wherein R¹ andR² are PG.
 20. The compound of claim 18, wherein R² is H.
 21. Thecompound of claim 18, wherein R¹ is H.
 22. The compound of claim 18,wherein R¹ is PG.
 23. The compound of claim 18, wherein R² is PG. 24.The compound of claim 18, wherein R³ and R⁴ are methyl.
 25. The compoundof claim 18, wherein PG is selected from the group consisting of FMOC,Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, andTs.
 26. The compound of claim 18, wherein PG is Boc.
 27. The compound ofclaim 18 having the formula


28. A process for preparing a compound of the formula I

comprising (a) contacting a compound of the formula A

with a compound of the formula B-14

in the presence of a base to provide a compound of the formula C

or (b) contacting a compound of the formula C with an inorganic base toprovide a compound of the formula D

or (c) contacting a compound of the formula D with an acid to provide acompound of the formula E

or (d) contacting a compound of the formula E with a base in thepresence of a phosphinate reagent to provide the compound of the formulaI.
 29. A process for preparing a compound of the formula B

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; comprising (a) contacting acompound of the formula B-1

wherein R⁴ is C₁-C₄ alkyl; with a compound of the formula B-2R

wherein R³ is C₁-C₄ alkyl, and PG is selected from the group consistingof FMOC, Boc, Cbz, Ac, trifluoroacetyl, phthalimide, Bn, trityl,benzylidene, and Ts; in the presence of an azodicarboxylate reagent anda phosphine reagent to provide a compound of the formula B-3

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; or (b) contacting a compoundof the formula B-3

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; with(R)-2-methyl-2-propanesulfinamide to provide a compound of the formulaB-5

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; or (c) contacting a compoundof the formula B-5

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; with a reducing agent toprovide a compound of the formula B-6

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; or (d) contacting a compoundof the formula B-6

wherein PG is selected from the group consisting of FMOC, Boc, Cbz, Ac,trifluoroacetyl, phthalimide, Bn, trityl, benzylidene, and Ts; and R³and R⁴ are each independently C₁-C₄ alkyl; with an iodine reagent toprovide a compound of the formula B. 30.-32. (canceled)